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1.
Cell ; 186(4): 877-891.e14, 2023 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-36708705

RESUMEN

We introduce BacDrop, a highly scalable technology for bacterial single-cell RNA sequencing that has overcome many challenges hindering the development of scRNA-seq in bacteria. BacDrop can be applied to thousands to millions of cells from both gram-negative and gram-positive species. It features universal ribosomal RNA depletion and combinatorial barcodes that enable multiplexing and massively parallel sequencing. We applied BacDrop to study Klebsiella pneumoniae clinical isolates and to elucidate their heterogeneous responses to antibiotic stress. In an unperturbed population presumed to be homogeneous, we found within-population heterogeneity largely driven by the expression of mobile genetic elements that promote the evolution of antibiotic resistance. Under antibiotic perturbation, BacDrop revealed transcriptionally distinct subpopulations associated with different phenotypic outcomes including antibiotic persistence. BacDrop thus can capture cellular states that cannot be detected by bulk RNA-seq, which will unlock new microbiological insights into bacterial responses to perturbations and larger bacterial communities such as the microbiome.


Asunto(s)
Perfilación de la Expresión Génica , Análisis de Expresión Génica de una Sola Célula , Análisis de Secuencia de ARN , RNA-Seq , Bacterias/genética , Análisis de la Célula Individual
2.
Clin Infect Dis ; 78(1): 31-39, 2024 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-37633257

RESUMEN

BACKGROUND: The clinical and microbial factors associated with Klebsiella pneumoniae bloodstream infections (BSIs) are not well characterized. Prior studies have focused on highly resistant or hypervirulent isolates, limiting our understanding of K. pneumoniae strains that commonly cause BSI. We performed a record review and whole-genome sequencing to investigate the clinical characteristics, bacterial diversity, determinants of antimicrobial resistance, and risk factors for in-hospital death in a cohort of patients with K. pneumoniae BSI. METHODS: We identified 562 patients at Massachusetts General Hospital with K. pneumoniae BSIs between 2016 and 2022. We collected data on comorbid conditions, infection source, clinical outcomes, and antibiotic resistance and performed whole-genome sequencing on 108 sequential BSI isolates from 2021 to 2022. RESULTS: Intra-abdominal infection was the most common source of infection accounting for 34% of all BSIs. A respiratory tract source accounted for 6% of BSIs but was associated with a higher in-hospital mortality rate (adjusted odds ratio, 5.4 [95% confidence interval, 2.2-12.8]; P < .001 for comparison with other sources). Resistance to the first antibiotic prescribed was also associated with a higher risk of death (adjusted odds ratio, 5.2 [95% confidence interval, 2.2-12.4]; P < .001). BSI isolates were genetically diverse, and no clusters of epidemiologically and genetically linked cases were observed. Virulence factors associated with invasiveness were observed at a low prevalence, although an unexpected association between O-antigen type and the source of infection was found. CONCLUSIONS: These observations demonstrate the versatility of K. pneumoniae as an opportunistic pathogen and highlight the need for new approaches for surveillance and the rapid identification of patients with invasive antimicrobial-resistant K. pneumoniae infection.


Asunto(s)
Bacteriemia , Infección Hospitalaria , Infecciones por Klebsiella , Sepsis , Humanos , Klebsiella pneumoniae , Infección Hospitalaria/epidemiología , Mortalidad Hospitalaria , Bacteriemia/microbiología , Infecciones por Klebsiella/microbiología , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Sepsis/tratamiento farmacológico , Genómica
3.
Respir Res ; 24(1): 23, 2023 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-36681813

RESUMEN

BACKGROUND: Low-dose spiral computed tomography (LDCT) may not lead to a clear treatment path when small to intermediate-sized lung nodules are identified. We have combined flow cytometry and machine learning to develop a sputum-based test (CyPath Lung) that can assist physicians in decision-making in such cases. METHODS: Single cell suspensions prepared from induced sputum samples collected over three consecutive days were labeled with a viability dye to exclude dead cells, antibodies to distinguish cell types, and a porphyrin to label cancer-associated cells. The labeled cell suspension was run on a flow cytometer and the data collected. An analysis pipeline combining automated flow cytometry data processing with machine learning was developed to distinguish cancer from non-cancer samples from 150 patients at high risk of whom 28 had lung cancer. Flow data and patient features were evaluated to identify predictors of lung cancer. Random training and test sets were chosen to evaluate predictive variables iteratively until a robust model was identified. The final model was tested on a second, independent group of 32 samples, including six samples from patients diagnosed with lung cancer. RESULTS: Automated analysis combined with machine learning resulted in a predictive model that achieved an area under the ROC curve (AUC) of 0.89 (95% CI 0.83-0.89). The sensitivity and specificity were 82% and 88%, respectively, and the negative and positive predictive values 96% and 61%, respectively. Importantly, the test was 92% sensitive and 87% specific in cases when nodules were < 20 mm (AUC of 0.94; 95% CI 0.89-0.99). Testing of the model on an independent second set of samples showed an AUC of 0.85 (95% CI 0.71-0.98) with an 83% sensitivity, 77% specificity, 95% negative predictive value and 45% positive predictive value. The model is robust to differences in sample processing and disease state. CONCLUSION: CyPath Lung correctly classifies samples as cancer or non-cancer with high accuracy, including from participants at different disease stages and with nodules < 20 mm in diameter. This test is intended for use after lung cancer screening to improve early-stage lung cancer diagnosis. Trial registration ClinicalTrials.gov ID: NCT03457415; March 7, 2018.


Asunto(s)
Neoplasias Pulmonares , Humanos , Detección Precoz del Cáncer/métodos , Citometría de Flujo , Pulmón , Neoplasias Pulmonares/diagnóstico por imagen , Aprendizaje Automático , Esputo
4.
Med Mycol ; 60(9)2022 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-36002024

RESUMEN

Invasive fungal infections are increasingly common and carry high morbidity and mortality, yet fungal diagnostics lag behind bacterial diagnostics in rapidly identifying the causal pathogen. We previously devised a fluorescent hybridization-based assay to identify bacteria within hours directly from blood culture bottles without subculture, called phylogeny-informed rRNA-based strain identification (Phirst-ID). Here, we adapt this approach to unambiguously identify 11 common pathogenic Candida species, including C. auris, with 100% accuracy from laboratory culture (33 of 33 strains in a reference panel, plus 33 of 33 additional isolates tested in a validation panel). In a pilot study on 62 consecutive positive clinical blood cultures from two hospitals that showed yeast on Gram stain, Candida Phirst-ID matched the clinical laboratory result for 58 of 59 specimens represented in the 11-species reference panel, without misclassifying the 3 off-panel species. It also detected mixed Candida species in 2 of these 62 specimens, including the one discordant classification, that were not identified by standard clinical microbiology workflows; in each case the presence of both species was validated by both clinical and experimental data. Finally, in three specimens that grew both bacteria and yeast, we paired our prior bacterial probeset with this new Candida probeset to detect both pathogen types using Phirst-ID. This simple, robust assay can provide accurate Candida identification within hours directly from blood culture bottles, and the conceptual approach holds promise for pan-microbial identification in a single workflow. LAY SUMMARY: Candida bloodstream infections cause considerable morbidity and mortality, yet slow diagnostics delay recognition, worsening patient outcomes. We develop and validate a novel molecular approach to accurately identify Candida species directly from blood culture one day faster than standard workflows.


Asunto(s)
Candida , Candidiasis , Animales , Cultivo de Sangre/veterinaria , Candidiasis/microbiología , Candidiasis/veterinaria , Proyectos Piloto , Saccharomyces cerevisiae
5.
J Infect Dis ; 224(10): 1658-1663, 2021 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-34255846

RESUMEN

Transmission of coronavirus disease 2019 (COVID-19) from people without symptoms confounds societal mitigation strategies. From April to June 2020, we tested nasopharyngeal swabs by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) from 15 514 staff and 16 966 residents of nursing homes and assisted living facilities in Massachusetts. Cycle threshold (Ct) distributions were very similar between populations with (n = 739) and without (n = 2179) symptoms at the time of sampling (mean Ct, 25.7 vs 26.4; ranges 12-38). However, as local cases waned, those without symptoms shifted towards higher Ct. With such similar viral load distributions, existing testing modalities should perform comparably regardless of symptoms, contingent upon time since infection.


Asunto(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Estudios Transversales , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga Viral
6.
Artículo en Inglés | MEDLINE | ID: mdl-33846128

RESUMEN

Current growth-based antibiotic susceptibility testing (AST) is too slow to guide early therapy. We previously developed a diagnostic approach that quantifies antibiotic-induced transcriptional signatures to distinguish susceptible from resistant isolates, providing phenotypic AST 24 to 36 h faster than current methods. Here, we show that 10 transcripts optimized for AST of one fluoroquinolone, aminoglycoside, or beta-lactam reflect susceptibility when the organism is exposed to other members of that class. This finding will streamline development and implementation of this strategy, facilitating efficient antibiotic deployment.


Asunto(s)
Antibacterianos , beta-Lactamas , Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana
7.
N Engl J Med ; 386(19): 1867-1868, 2022 05 12.
Artículo en Inglés | MEDLINE | ID: mdl-35443125
9.
Artículo en Inglés | MEDLINE | ID: mdl-31138575

RESUMEN

Quantitative assessment of antibiotic-responsive RNA transcripts holds promise for a rapid point-of-care (POC) diagnostic tool for antimicrobial susceptibility testing. These assays aim to distinguish susceptible and resistant isolates by transcriptional differences upon drug exposure. However, an often-overlooked dimension of designing these tests is that the genetic diversity within a species may yield differential transcriptional regulation independent of resistance phenotype. Here, we use a phylogenetically diverse panel of Neisseria gonorrhoeae and transcriptome profiling coupled with reverse transcription-quantitative PCR to test this hypothesis, to identify azithromycin responsive transcripts and evaluate their potential diagnostic value, and to evaluate previously reported diagnostic markers for ciprofloxacin resistance (porB and rpmB). Transcriptome profiling confirmed evidence of genetic distance and population structure impacting transcriptional response to azithromycin. Taking this into account, we found azithromycin-responsive transcripts overrepresented in susceptible strains compared to resistant strains and selected four candidate diagnostic transcripts (rpsO, rplN, omp3, and NGO1079) that were the most significantly differentially regulated between phenotypes across drug exposure. RNA signatures for these markers categorically predicted resistance in 19/20 cases, with the one incorrect categorical assignment for an isolate at the threshold of reduced susceptibility. Finally, we found that porB and rpmB expression were not uniformly diagnostic of ciprofloxacin resistance in a panel of isolates with unbiased phylogenetic sampling. Overall, our results suggest that RNA signatures as a diagnostic tool are promising for future POC diagnostics; however, development and testing should consider representative genetic diversity of the target pathogen.


Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genética , ARN/genética , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Fenotipo , Filogenia
10.
Nat Methods ; 12(4): 323-5, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25730492

RESUMEN

Although RNA-seq is a powerful tool, the considerable time and cost associated with library construction has limited its utilization for various applications. RNAtag-Seq, an approach to generate multiple RNA-seq libraries in a single reaction, lowers time and cost per sample, and it produces data on prokaryotic and eukaryotic samples that are comparable to those generated by traditional strand-specific RNA-seq approaches.


Asunto(s)
Secuencia de Bases , Biblioteca de Genes , Análisis de Secuencia de ARN/métodos , Bacterias/genética , Perfilación de la Expresión Génica/normas , Análisis de Secuencia de ARN/economía , Análisis de Secuencia de ARN/normas , Factores de Tiempo
11.
J Am Soc Nephrol ; 32(2): 519-520, 2021 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-36734843
16.
Nat Microbiol ; 9(1): 95-107, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38168615

RESUMEN

The host type I interferon (IFN) pathway is a major signature of inflammation induced by the human fungal pathogen, Candida albicans. However, the molecular mechanism for activating this pathway in the host defence against C. albicans remains unknown. Here we reveal that mice lacking cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway components had improved survival following an intravenous challenge by C. albicans. Biofilm-associated C. albicans DNA packaged in extracellular vesicles triggers the cGAS-STING pathway as determined by induction of interferon-stimulated genes, IFNß production, and phosphorylation of IFN regulatory factor 3 and TANK-binding kinase 1. Extracellular vesicle-induced activation of type I IFNs was independent of the Dectin-1/Card9 pathway and did not require toll-like receptor 9. Single nucleotide polymorphisms in cGAS and STING potently altered inflammatory cytokine production in human monocytes challenged by C. albicans. These studies provide insights into the early innate immune response induced by a clinically significant fungal pathogen.


Asunto(s)
Candidiasis , Interferón Tipo I , Animales , Ratones , Candida albicans/patogenicidad , Proteínas Adaptadoras de Señalización CARD/metabolismo , Inmunidad Innata , Interferón Tipo I/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Transducción de Señal , Candidiasis/metabolismo , Candidiasis/patología
17.
Nat Genet ; 25(4): 440-3, 2000 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-10932191

RESUMEN

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.


Asunto(s)
Genes/fisiología , Genoma , Mutagénesis/genética , Animales , Animales Recién Nacidos , Mapeo Cromosómico , Cruzamientos Genéticos , Criopreservación , Etilnitrosourea/farmacología , Femenino , Fertilización In Vitro , Genes/efectos de los fármacos , Genes/genética , Pruebas Hematológicas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Actividad Motora/genética , Mutagénesis/efectos de los fármacos , Mutágenos/farmacología , Mutación , Fenotipo , Factores de Tiempo , Destete
18.
bioRxiv ; 2023 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-37292717

RESUMEN

Carbapenem-resistant Enterobacterales (CRE) are important pathogens that can develop resistance via multiple molecular mechanisms, including hydrolysis or reduced antibiotic influx. Identifying these mechanisms can improve pathogen surveillance, infection control, and patient care. We investigated how resistance mechanisms influence the carbapenem inoculum effect (IE), a phenomenon where inoculum size affects antimicrobial susceptibility testing (AST). We demonstrated that seven different carbapenemases impart a meropenem IE in Escherichia coli. Across 110 clinical CRE isolates, the carbapenem IE strictly depended on resistance mechanism: all carbapenemase-producing CRE (CP-CRE) exhibited a strong IE, whereas porin-deficient CRE displayed none. Concerningly, 50% and 24% of CP-CRE isolates changed susceptibility classification to meropenem and ertapenem, respectively, across the allowable inoculum range in clinical guidelines. The meropenem IE, and the ratio of ertapenem to meropenem minimal inhibitory concentration (MIC) at standard inoculum, reliably identified CP-CRE. Understanding how resistance mechanisms affect AST could improve diagnosis and guide therapies for CRE infections.

19.
ACS Infect Dis ; 9(1): 111-121, 2023 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-36519726

RESUMEN

Using genome mining and heterologous expression, we report the discovery and production of a new antimicrobial lasso peptide from species related to the Enterobacter cloacae complex. Using NMR and mass spectrometric analysis, we show that this lasso peptide, named cloacaenodin, employs a threaded lasso fold which imparts proteolytic resistance that its unthreaded counterpart lacks. Cloacaenodin has selective, low micromolar, antimicrobial activity against species related to the E. cloacae complex, including species implicated in nosocomial infections and against clinical isolates of carbapenem-resistant Enterobacterales. We further used site-directed mutagenesis to probe the importance of specific residues to the peptide's biosynthesis, stability, and bioactivity.


Asunto(s)
Antibacterianos , Enterobacter , Enterobacter/genética , Antibacterianos/farmacología , Antibacterianos/química , Péptidos Antimicrobianos , Carbapenémicos , Péptidos/farmacología , Péptidos/química
20.
Open Forum Infect Dis ; 9(12): ofac621, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-36544861

RESUMEN

We conducted an ecological analysis of the dynamics of Delta and Omicron establishment and dominance in US states. Omicron became the dominant circulating variant later in states with higher population-level immunity. By contrast, population immunity did not impact the maximum rate of takeover by Delta or Omicron from prior variants.

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