RESUMO
There are no existing affordable diagnostics for sensitive, rapid, and on-site detection of pathogens in milk. To this end, an on-site colorimetric-based sustainable assay has been developed and optimized using an L16 (54) Taguchi design to obtain results in hours without PCR amplification. To determine the level of Escherichia coli (E. coli) contamination, after induction with 150 µL of breast milk, the B-Per bacterial protein extraction kit was added to a solution containing an alginate-based microcapsule assay. Within this 3 mm spherical novel sensor design, X-Gal (5-Bromo-4-Chloro-3-Indolyl ß-d-Galactopyranoside) was entrapped at a concentration of 2 mg/mL. The outward diffusing X-Gal was cleaved by ß-galactosidase from E. coli and dimerized in the solution to yield a blue color after incubation at 40 °C. Color intensity was correlated with the level of E. coli contamination using a categorical scale. After an 8 h incubation period, a continuous imaging scale based on intensity normalization was used to determine a binary lower limit of detection (LOD), which corresponded to 102 colony forming unit per mL (CFU/mL) and above. The cost of the overall assay was estimated to be $0.81 per sample, well under the $3 benchmark for state-of-the-art immune-based test kits for pathogen detection in biofluids. Considering the reported binary LOD cutoff of 102 CFU/mL and above, this proposed hydrogel-based assay is suited to meet global requirements for screening breast milk or milk for pathogenic organisms of 104 CFU/mL, with a percentage of false positives to be determined in future efforts.
Assuntos
Alginatos/química , Técnicas Biossensoriais/métodos , Escherichia coli/isolamento & purificação , Leite Humano/microbiologia , Catálise , Humanos , Limite de Detecção , Padrões de Referência , Razão Sinal-RuídoRESUMO
BACKGROUND: Treatment of prostate cancer (PCa) may be improved by identifying biological mechanisms of tumor growth that directly impact clinical disease progression. We investigated whether genes associated with a highly tumorigenic, drug resistant, progenitor phenotype impact PCa biology and recurrence. METHODS: Radical prostatectomy (RP) specimens (±disease recurrence, N = 276) were analyzed by qRT-PCR to quantify expression of genes associated with self-renewal, drug resistance, and tumorigenicity in prior studies. Associations between gene expression and PCa recurrence were confirmed by bootstrap internal validation and by external validation in independent cohorts (total N = 675) and in silico. siRNA knockdown and lentiviral overexpression were used to determine the effect of gene expression on PCa invasion, proliferation, and tumor growth. RESULTS: Four candidate genes were differentially expressed in PCa recurrence. Of these, low AXIN2 expression was internally validated in the discovery cohort. Validation in external cohorts and in silico demonstrated that low AXIN2 was independently associated with more aggressive PCa, biochemical recurrence, and metastasis-free survival after RP. Functionally, siRNA-mediated depletion of AXIN2 significantly increased invasiveness, proliferation, and tumor growth. Conversely, ectopic overexpression of AXIN2 significantly reduced invasiveness, proliferation, and tumor growth. CONCLUSIONS: Low AXIN2 expression was associated with PCa recurrence after RP in our test population as well as in external validation cohorts, and its expression levels in PCa cells significantly impacted invasiveness, proliferation, and tumor growth. Given these novel roles, further study of AXIN2 in PCa may yield promising new predictive and therapeutic strategies.
Assuntos
Proteína Axina , Próstata , Prostatectomia/métodos , Neoplasias da Próstata , Idoso , Proteína Axina/análise , Proteína Axina/genética , Biomarcadores , Humanos , Masculino , Gradação de Tumores , Invasividade Neoplásica , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Prognóstico , Próstata/patologia , Próstata/cirurgia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Medição de RiscoRESUMO
Most Escherichia coli isolates from humans do not utilize D-sucrose as a substrate for fermentation or growth. Previous work has shown that the Csc pathway allows some E. coli to utilize sucrose for slow growth, and this pathway has been engineered in E. coli W strains to enhance use of sucrose as a feedstock for industrial applications. An alternative sucrose utilization pathway, Scr, was first identified in Klebsiella pneumoniae and has been reported in some E. coli and Salmonella enterica isolates. We show here that the Scr pathway is native to an important subset of E. coli phylogroup B2 lineages that lack the Csc pathway but grow rapidly on sucrose. Laboratory E. coli strains derived from MG1655 (phylogroup A, ST10) are unable to utilize sucrose and lack the scr and csc genes, but a recombinant plasmid-borne scr locus enables rapid growth on and fermentation of sucrose. Genome analyses of Enterobacteriaceae indicate that the scr locus is widespread in other Enterobacteriaceae; including Enterobacter and Klebsiella species, and some Citrobacter and Proteus species. In contrast, the Csc pathway is limited mostly to E. coli, some Shigella species (in which csc loci are rendered non-functional by various mutations), and Citrobacter freundii. The more efficient Scr pathway likely has greater potential than the Csc pathway for bioindustrial applications of E. coli and other Enterobacteriaceae using sucrose as a feedstock.
RESUMO
Xenorhabdus species are bacterial symbionts of entomopathogenic Steinernema nematodes, in which they produce diverse secondary metabolites implicated in pathogenesis. To expand resources for natural product prospecting and exploration of host-symbiont-pathogen relationships, the genomes of Xenorhabdus cabanillasi, Xenorhabdus ehlersii, Xenorhabdus japonica, Xenorhabdus koppenhoeferii, and Xenorhabdus mauleonii were analyzed.
RESUMO
Whole genome sequencing (WGS) of clinical bacterial isolates has the potential to transform the fields of diagnostics and public health. To realize this potential, bioinformatic software that reports identification results needs to be developed that meets the quality standards of a diagnostic test. We developed GAMBIT (Genomic Approximation Method for Bacterial Identification and Tracking) using k-mer based strategies for identification of bacteria based on WGS reads. GAMBIT incorporates this algorithm with a highly curated searchable database of 48,224 genomes. Herein, we describe validation of the scoring methodology, parameter robustness, establishment of confidence thresholds and the curation of the reference database. We assessed GAMBIT by way of validation studies when it was deployed as a laboratory-developed test in two public health laboratories. This method greatly reduces or eliminates false identifications which are often detrimental in a clinical setting.
Assuntos
Bactérias , Genômica , Sequenciamento Completo do Genoma/métodos , Bactérias/genética , Software , Biologia Computacional , Genoma BacterianoRESUMO
Billions of microbes sculpt the gut ecosystem, affecting physiology. Since endurance athletes' performance is often physiology-limited, understanding the composition and interactions within athletes' gut microbiota could improve performance. Individual studies describe differences in the relative abundance of bacterial taxa in endurance athletes, suggesting the existence of an "endurance microbiota", yet the taxa identified are mostly non-overlapping. To narrow down the source of this variation, we created a bioinformatics workflow and reanalyzed fecal microbiota from four 16S rRNA gene sequence datasets associated with endurance athletes and controls, examining diversity, relative abundance, correlations, and association networks. There were no significant differences in alpha diversity among all datasets and only one out of four datasets showed a significant overall difference in bacterial community abundance. When bacteria were examined individually, there were no genera with significantly different relative abundance in all four datasets. Two genera were significantly different in two datasets (Veillonella and Romboutsia). No changes in correlated abundances were consistent across datasets. A power analysis using the variance in relative abundance detected in each dataset indicated that much larger sample sizes will be necessary to detect a modest difference in relative abundance especially given the multitude of covariates. Our analysis confirms several challenges when comparing microbiota in general, and indicates that microbes consistently or universally associated with human endurance remain elusive.
RESUMO
Recent advances in understanding bacterial cell-cycle regulation suggest circuit control mechanisms that operate analogously to those in the eukaryotic cell cycle.
Assuntos
Caulobacter crescentus/citologia , Ciclo Celular/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Regulação Bacteriana da Expressão Gênica , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Staphylococcus saprophyticus is a significant cause of urinary tract infections in younger women, but it has been understudied at the genomic level. We report genome sequences of six S. saprophyticus isolates obtained from female patients who presented with urinary tract infection symptoms at a college health center in 2019.
RESUMO
The evolution and propagation of antibiotic resistance by bacterial pathogens are significant threats to global public health. Contemporary DNA sequencing tools were applied here to gain insight into carriage of antibiotic resistance genes in Escherichia coli, a ubiquitous commensal bacterium in the gut microbiome in humans and many animals, and a common pathogen. Draft genome sequences generated for a collection of 101 E. coli strains isolated from healthy undergraduate students showed that horizontally acquired antibiotic resistance genes accounted for most resistance phenotypes, the primary exception being resistance to quinolones due to chromosomal mutations. A subset of 29 diverse isolates carrying acquired resistance genes and 21 control isolates lacking such genes were further subjected to long-read DNA sequencing to enable complete or nearly complete genome assembly. Acquired resistance genes primarily resided on F plasmids (101/153 [67%]), with smaller numbers on chromosomes (30/153 [20%]), IncI complex plasmids (15/153 [10%]), and small mobilizable plasmids (5/153 [3%]). Nearly all resistance genes were found in the context of known transposable elements. Very few structurally conserved plasmids with antibiotic resistance genes were identified, with the exception of an â¼90-kb F plasmid in sequence type 1193 (ST1193) isolates that appears to serve as a platform for resistance genes and may have virulence-related functions as well. Carriage of antibiotic resistance genes on transposable elements and mobile plasmids in commensal E. coli renders the resistome highly dynamic.IMPORTANCE Rising antibiotic resistance in human-associated bacterial pathogens is a serious threat to our ability to treat many infectious diseases. It is critical to understand how acquired resistance genes move in and through bacteria associated with humans, particularly for species such as Escherichia coli that are very common in the human gut but can also be dangerous pathogens. This work combined two distinct DNA sequencing approaches to allow us to explore the genomes of E. coli from college students to show that the antibiotic resistance genes these bacteria have acquired are usually carried on a specific type of plasmid that is naturally transferrable to other E. coli, and likely to other related bacteria.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Fator F/genética , Simbiose , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/microbiologia , Transferência Genética Horizontal , Genoma Bacteriano , Humanos , Testes de Sensibilidade Microbiana , Análise de Sequência de DNA , Adulto JovemRESUMO
Sensing of magnetic fields by living organisms -- magnetosensing -- is best understood in magnetotactic bacteria. Recently work has provided new insight into the biogenesis of bacterial magnetosomes, and links these organelles to a newly recognized prokaryotic cytoskeletal filament which organizes magnetosomes into a sensory structure capable of aligning cells with the geomagnetic field.
Assuntos
Magnetospirillum/fisiologia , Animais , Citoesqueleto , Óxido Ferroso-Férrico , Magnetospirillum/ultraestruturaRESUMO
Cellular senescence, even in the presence of abundant nutrients, has now been demonstrated in several microbes, including most recently the bacterium Escherichia coli, suggesting that it may be a universal adaptive response to the inevitable damage to cell constituents that accumulates with time.
Assuntos
Adaptação Fisiológica , Fenômenos Fisiológicos Bacterianos , Divisão Celular/fisiologia , Fatores de TempoRESUMO
BACKGROUND: Few clinical studies have investigated the association between neutrophil-lymphocyte ratio (NLR) and treatment with cetuximab-based chemotherapy in metastatic colorectal cancer (mCRC). The NLR may reflect immune cells modulating specific cytokine signals in the tumor microenvironment; however, which immune-related genes affect the NLR remain unclear. PATIENTS AND METHODS: In 77 patients with KRAS exon2 wild-type mCRC from prospective trials of first-line chemotherapy with cetuximab, expression levels of 354 immune-related genes were measured in tissue samples obtained from all patients by the HTG EdgeSeq Oncology Biomarker Panel. The association between the NLR and clinical outcomes was evaluated using the Spearman rank correlation coefficient. In addition, 2-sample t tests were performed to investigate which genes among the top 100 genes associated with survival had significantly different expression levels between the NLR-low and NLR-high groups among all measured genes. RESULTS: NLR data were available for 71 patients. The NLR was associated with progression-free survival and overall survival (r = -0.24; P = .040 and r = -0.29; P = .010, respectively). When stratified by the median value of the NLR, the Kaplan-Meier curve of NLR-low versus NLR-high differed significantly for both progression-free survival (median, 11.8 vs. 9.1 months; P = .036) and overall survival (median, 42.8 vs. 26.7 months; P = .029). The 2-sample t test revealed that the expression levels of the LYZ, TYMP, and CD68 genes differed significantly between the NLR-low and NLR-high groups (t test P-value < .005; false discovery rate P-value < .15). CONCLUSION: NLR is significantly associated with survival in patients with mCRC treated with first-line chemotherapy with cetuximab. Genes encoding for activities on macrophages may affect the NLR.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores/metabolismo , Neoplasias Colorretais/mortalidade , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Hepáticas/mortalidade , Linfócitos/patologia , Neutrófilos/patologia , Cetuximab/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Fluoruracila/administração & dosagem , Seguimentos , Humanos , Leucovorina/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Oxaliplatina/administração & dosagem , Prognóstico , Estudos Prospectivos , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Molecular differences in tumor locations may contribute to the sidedness-specific response to cetuximab in metastatic colorectal cancer (mCRC). We investigated genes associated with the response to cetuximab treatment depending on tumor sidedness. Our study included 77 patients with mCRC (13/63, right/left) with KRAS exon 2 wild-type tumors from phase II trials of first-line therapy with cetuximab. Expression levels of 2,551 genes were measured in tissue samples by HTG EdgeSeq Oncology Biomarker Panel. Univariate Cox regression analysis using log2 values of counts per million (CPM) was conducted in each sidedness to assess associations with clinical outcomes, and to define the optimal cut-off point for clinically significant genes. In addition, a gene set enrichment analysis (GSEA) was performed to identify significant gene pathways in each sidedness. Sixty-nine patients were assessable for gene expression data. Overexpression of BECN1 [log2(CPM) ≥ 6.8] was associated with favorable survival, regardless of tumor sidedness. High expression of NOTCH1 [log2(CPM) ≥ 7.5] predicted significantly longer progression-free survival (PFS; median 14.7 vs. 11.1 months, HR 0.43, P = 0.01) and overall survival (OS; median 42.8 vs. 26.5 months, HR 0.35, P = 0.01) in left side but not in right side. The GSEA showed that regulation of DNA replication gene set correlated with favorable survival in the left, whereas the subcellular component and leukocyte migration gene sets were associated with good survival in the right. In conclusion, genes contributing to the efficacy of cetuximab treatment may differ according to the sidedness in mCRC. NOTCH1 may potentially discriminate favorable responders to cetuximab in patients with left-sided tumors.
Assuntos
Cetuximab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Genes Neoplásicos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Metástase Neoplásica , Prognóstico , Receptor Notch1/metabolismoRESUMO
In the oligotrophic freshwater bacterium Caulobacter crescentus, D-xylose induces expression of over 50 genes, including the xyl operon, which encodes key enzymes for xylose metabolism. The promoter (P(xylX)) controlling expression of the xyl operon is widely used as a tool for inducible heterologous gene expression in C. crescentus. We show here that P(xylX) and at least one other promoter in the xylose regulon (P(xylE)) are controlled by the CC3065 (xylR) gene product, a LacI-type repressor. Electrophoretic gel mobility shift assays showed that operator binding by XylR is greatly reduced in the presence of D-xylose. The data support the hypothesis that there is a simple regulatory mechanism in which XylR obstructs xylose-inducible promoters in the absence of the sugar; the repressor is induced to release DNA upon binding D-xylose, thereby freeing the promoter for productive interaction with RNA polymerase. XylR also has an effect on glucose metabolism, as xylR mutants exhibit reduced expression of the Entner-Doudoroff operon and their ability to utilize glucose as a sole carbon and energy source is compromised.
Assuntos
Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/genética , Xilose/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Glucose/metabolismo , Regiões Operadoras Genéticas , Ligação Proteica , Proteínas Repressoras/metabolismoRESUMO
A genetic regulatory circuit recently described in the bacterium Caulobacter crescentus generates reciprocal oscillations in the abundance of two key transcription factors to control landmark events in the cell cycle.
Assuntos
Caulobacter crescentus/genética , Ciclo Celular/genética , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/genética , Relógios Biológicos/genética , Ciclo Celular/fisiologia , Replicação do DNA/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismoRESUMO
A Brazilian consortium has unveiled the genomic DNA sequence of the purple-pigmented bacterium Chromobacterium violaceum, a dominant component of the tropical soil microbiota. The sequence provides insight into the abundant potential of this organism for biotechnological and pharmaceutical applications.
Assuntos
Chromobacterium/genética , Regulação da Expressão Gênica , Genoma , Pigmentação/fisiologia , Microbiologia do Solo , Chromobacterium/fisiologia , IndóisRESUMO
The ParM ATPase from Escherichia coli plasmid R1 assembles into F-actin-like filaments which appear to push replicated copies of the plasmid to opposite ends of the cell, ensuring partitioning into daughter cells. Might bacterial chromosomes use a similar mitotic strategy for segregation?
Assuntos
Cromossomos Bacterianos , Escherichia coli/genética , PlasmídeosRESUMO
Biofilms--adherent bacterial communities embedded in a polymer matrix--are common in nature and can cause persistent human infections that are highly resistant to antibiotics. Recent work provides insight into how these communities develop and function, and offers clues to combating them.
Assuntos
Biofilmes/crescimento & desenvolvimento , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/patogenicidade , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Biofilmes/efeitos dos fármacos , Adesão Celular , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Projects designed to scan entire microbial genomes for essential genes have revealed a remarkably compact and conserved, but not universal, set of genes whose functions are necessary for survival or reproduction.
Assuntos
Bacillus subtilis/genética , Genômica/métodos , Genes Essenciais/genética , Genômica/tendênciasRESUMO
Extraintestinal pathogenic Escherichia coli (ExPEC) strains belonging to multilocus sequence type 95 (ST95) are globally distributed and a common cause of infections in humans and domestic fowl. ST95 isolates generally show a lower prevalence of acquired antimicrobial resistance than other pandemic ExPEC lineages. We took a genomic approach to identify factors that may underlie reduced resistance. We fully assembled genomes for four ST95 isolates representing the four major fimH-based lineages within ST95 and also analyzed draft-level genomes from another 82 ST95 isolates, largely from the western United States. The fully assembled genomes of antibiotic-resistant isolates carried resistance genes exclusively on large (>90-kb) IncFIB/IncFII plasmids. These replicons were common in the draft genomes as well, particularly in antibiotic-resistant isolates, but we also observed multiple instances of a smaller (8.3-kb) ampicillin resistance plasmid that had been previously identified in Salmonella enterica. Among ST95 isolates, pansusceptibility to antibiotics was significantly associated with the fimH6 lineage and the presence of homologs of the previously identified 114-kb IncFIB/IncFII plasmid pUTI89, both of which were also associated with reduced carriage of other plasmids. Potential mechanistic explanations for lineage- and plasmid-specific effects on the prevalence of antibiotic resistance within the ST95 group are discussed. IMPORTANCE Antibiotic resistance in bacterial pathogens is a major public health concern. This work was motivated by the observation that only a small proportion of ST95 isolates, a major pandemic lineage of extraintestinal pathogenic E. coli, have acquired antibiotic resistance, in contrast to many other pandemic lineages. Understanding bacterial genetic factors that may prevent acquisition of resistance could contribute to the development of new biological, medical, or public health strategies to reduce antibiotic-resistant infections.