RESUMEN

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

Asunto(s)

ADN Bacteriano , Microbiología de Alimentos , Leche , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Salmonella , Técnicas de Amplificación de Ácido Nucleico/métodos , Microbiología de Alimentos/métodos , Animales , Leche/microbiología , Salmonella/genética , Salmonella/aislamiento & purificación , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Enfermedades Transmitidas por los Alimentos/microbiología , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , Porcinos

RESUMEN

Foodborne pathogens continue to be a major health concern worldwide. Culture-dependent methodologies are still considered the gold standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor intensive. The implementation of real-time PCR has allowed to overcome these limitations, and even reduce the cost associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter, a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.

Asunto(s)

Microbiología de Alimentos , Enfermedades Transmitidas por los Alimentos , Listeria monocytogenes , Reacción en Cadena de la Polimerasa Multiplex , Salmonella , Listeria monocytogenes/genética , Listeria monocytogenes/aislamiento & purificación , Microbiología de Alimentos/métodos , Salmonella/genética , Salmonella/aislamiento & purificación , Reacción en Cadena de la Polimerasa Multiplex/métodos , Enfermedades Transmitidas por los Alimentos/microbiología , Enfermedades Transmitidas por los Alimentos/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Humanos , ADN Bacteriano/genética , ADN Bacteriano/análisis

RESUMEN

Foodborne pathogens remain a serious health issue in developed and developing countries. Safeness of food products has been assured for years with culture-based microbiological methods; however, these present several limitations such as turnaround time and extensive hands-on work, which have been typically address taking advantage of DNA-based methods such as real-time PCR (qPCR). These, and other similar techniques, are targeted assays, meaning that they are directed for the specific detection of one specific microbe. Even though reliable, this approach suffers from an important limitation that unless specific assays are design for every single pathogen potentially present, foods may be considered erroneously safe. To address this problem, next-generation sequencing (NGS) can be used as this is a nontargeted method; thus it has the capacity to detect every potential threat present. In this chapter, a protocol for the simultaneous detection and preliminary serotyping of Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Escherichia coli O157:H7 is described.

Asunto(s)

Microbiología de Alimentos , Enfermedades Transmitidas por los Alimentos , Secuenciación de Nucleótidos de Alto Rendimiento , Listeria monocytogenes , Microbiología de Alimentos/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Enfermedades Transmitidas por los Alimentos/microbiología , Enfermedades Transmitidas por los Alimentos/diagnóstico , Listeria monocytogenes/aislamiento & purificación , Listeria monocytogenes/genética , Escherichia coli O157/aislamiento & purificación , Escherichia coli O157/genética , Humanos , Serotipificación/métodos , ADN Bacteriano/genética , ADN Bacteriano/análisis , Salmonella typhimurium/aislamiento & purificación , Salmonella typhimurium/genética

RESUMEN

The standardization of the microbiome sequencing of poultry rinsates is essential for generating comparable microbial composition data among poultry processing facilities if this technology is to be adopted by the industry. Samples must first be acquired, DNA must be extracted, and libraries must be constructed. In order to proceed to library sequencing, the samples should meet quality control standards. Finally, data must be analyzed using computer bioinformatics pipelines. This data can subsequently be incorporated into more advanced computer algorithms for risk assessment. Ultimately, *a uniform sequencing pipeline will enable both the government regulatory agencies and the poultry industry to identify potential weaknesses in food safety.This chapter presents the different steps for monitoring the population dynamics of the microbiome in poultry processing using 16S rDNA sequencing.

Asunto(s)

Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Microbiota , Aves de Corral , ARN Ribosómico 16S , Animales , ARN Ribosómico 16S/genética , Aves de Corral/microbiología , Microbiota/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Biología Computacional/métodos , ADN Bacteriano/genética

RESUMEN

A high cell-surface hydrophobic bacterium, strain A18T, was isolated from a waste digestion system in Chaozhou, China. Cells of strain A18T were Gram-stain-positive, aerobic, non-spore-forming, non-motile, and rod-shaped. Phylogenetic analyses based on the 16S rRNA gene showed that strain A18T shared less than 94.2% sequence similarity to all validated species in the family Chitinophagaceae, and formed a distinct lineage close to genera Niabella and Terrimonas in the neighbor-joining tree, indicating that strain A18T is a novel species. Genome-based phylogenetic analyses revealed that strain A18T is affiliated to the genus Niabella. The cellular components, including iso-C15:0 and iso-C15:1 G as the major fatty acids, menaquinone-7 as the respiratory quinone and a DNA G + C content of 40.54% supported strain A18T as a member of the genus Niabella. However, the physiological and biochemical properties, such as enzyme activities, carbon source utilization and C18:0 3-OH as another major fatty acids, distinguished strain A18T from its close related species. Therefore, the name Niabella digestorum sp. nov. is proposed for this novel species. The type strain is A18T (= GDMCC 1.3242 T = KCTC 92386 T).

Asunto(s)

Composición de Base , ADN Bacteriano , Ácidos Grasos , Filogenia , ARN Ribosómico 16S , ARN Ribosómico 16S/genética , Ácidos Grasos/metabolismo , ADN Bacteriano/genética , China , Técnicas de Tipificación Bacteriana , Interacciones Hidrofóbicas e Hidrofílicas , Bacteroidetes/genética , Bacteroidetes/clasificación , Bacteroidetes/aislamiento & purificación , Bacteroidetes/metabolismo , Análisis de Secuencia de ADN , Vitamina K 2/metabolismo , Vitamina K 2/análisis , Vitamina K 2/análogos & derivados

RESUMEN

The 16S rRNA gene of Thermobacterium salinum TK19130T had the highest sequence similarity to that of Luteirhabdus pelagi A3-108T (99.7%). Phylogeny of 16S rRNA gene and whole genome sequences indicated that T. salinum TK19130T and L. pelagi A3-108T are closely related, and represented an independent clade. Whole genome comparisons showed that T. salinum TK19130T and L. pelagi A3-108T shared average amino acid identity of 95.3%, indicating they could be merged into the same genus. The digital DNA-DNA hybridization and average nucleotide identity values between T. salinum TK19130T and L. pelagi A3-108T were 52.5 and 93.3%, respectively. These values were below the recommended threshold values of prokaryotic species delineation. Thus, based on the principle of priority, we proposed the transfer of Thermobacterium salinum Chen et al. 2023 to the genus Luteirhabdus Ren et al. 2022 as Luteirhabdus salina comb. nov.

Asunto(s)

ADN Bacteriano , Hibridación de Ácido Nucleico , Filogenia , ARN Ribosómico 16S , Análisis de Secuencia de ADN , ARN Ribosómico 16S/genética , ADN Bacteriano/genética , Técnicas de Tipificación Bacteriana , Genoma Bacteriano , Secuenciación Completa del Genoma

RESUMEN

A Gram-stain-negative, short rod-shaped strain, MDT2-1-1T, was isolated from cryoconite samples collected from the Midui glacier in Tibet, China. It grew aerobically from 7 to 40 °C, within a pH range of 6.0-10.0, and in NaCl concentration of 0 to 1.0% (w/v). The pairwise 16S rRNA gene sequence similarity, average nucleotide identity and digital DNA-DNA hybridization values between strains MDT2-1-1T and Sabulicella rubraurantiaca SYSU D01096T were 99.4%, 89.7% and 38.9%, respectively. Considering the results from phylogeny, phenotypic and genotypic data, strain MDT2-1-1T (=CGMCC 1.11170T = NBRC 110485T) was suggested to represent a novel species of the genus Sabulicella, for which the name Sabulicella glaciei sp. nov. is proposed. Furthermore, based on the phylogenomic analysis, it is recommended that Roseomonas rubea, Roseomonas ponticola and Roseomonas oleicola be reclassified as Neoroseomonas rubea comb. nov., Falsiroseomonas ponticola comb. nov. and Falsiroseomonas oleicola comb. nov., respectively. Considering the illegitimate status of the genera names Pararoseomonas and Pseudoroseomonas, the species within the genera Pararoseomonas and Pseudoroseomonas should be transferred to Muricoccus and Teichococcus, respectively. Therefore, we proposed the following new combinations: Muricoccus aeriglobus comb. nov., Muricoccus aerilatus comb. nov., Muricoccus harenae comb. nov., Muricoccus nepalensis comb. nov., Muricoccus pecuniae comb. nov., Muricoccus radiodurans comb. nov., Muricoccus vinaceus comb. nov., Teichococcus aerofrigidensis comb. nov., Teichococcus aerophilus comb. nov., Teichococcus aestuarii comb. nov., Teichococcus cervicalis comb. nov., Teichococcus coralli comb. nov., Teichococcus deserti comb. nov., Teichococcus globiformis comb. nov., Teichococcus hibiscisoli comb. nov., Teichococcus musae comb. nov., Teichococcus oryzae comb. nov., Teichococcus rhizosphaerae comb. nov., Teichococcus ruber comb. nov., Teichococcus suffuscus comb. nov., Teichococcus vastitatis comb. nov., and Teichococcus wenyumeiae comb. nov.

Asunto(s)

Técnicas de Tipificación Bacteriana , ADN Bacteriano , Hibridación de Ácido Nucleico , Filogenia , ARN Ribosómico 16S , ARN Ribosómico 16S/genética , ADN Bacteriano/genética , Ácidos Grasos/análisis , Tibet , Análisis de Secuencia de ADN , Composición de Base , Methylobacteriaceae/clasificación , Methylobacteriaceae/genética , Methylobacteriaceae/aislamiento & purificación , Methylobacteriaceae/fisiología

RESUMEN

BACKGROUND: Next-generation sequencing (NGS) approaches have revolutionized gut microbiome research and can provide strain-level resolution, but these techniques have limitations in that they are only semi-quantitative, suffer from high detection limits, and generate data that is compositional. The present study aimed to systematically compare quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for the absolute quantification of Limosilactobacillus reuteri strains in human fecal samples and to develop an optimized protocol for the absolute quantification of bacterial strains in fecal samples. RESULTS: Using strain-specific PCR primers for L. reuteri 17938, ddPCR showed slightly better reproducibility, but qPCR was almost as reproducible and showed comparable sensitivity (limit of detection [LOD] around 104 cells/g feces) and linearity (R2 > 0.98) when kit-based DNA isolation methods were used. qPCR further had a wider dynamic range and is cheaper and faster. Based on these findings, we conclude that qPCR has advantages over ddPCR for the absolute quantification of bacterial strains in fecal samples. We provide an optimized and easy-to-follow step-by-step protocol for the design of strain-specific qPCR assays, starting from primer design from genome sequences to the calibration of the PCR system. Validation of this protocol to design PCR assays for two L. reuteri strains, PB-W1 and DSM 20016 T, resulted in a highly accurate qPCR with a detection limit in spiked fecal samples of around 103 cells/g feces. Applying our strain-specific qPCR assays to fecal samples collected from human subjects who received live L. reuteri PB-W1 or DSM 20016 T during a human trial demonstrated a highly accurate quantification and sensitive detection of these two strains, with a much lower LOD and a broader dynamic range compared to NGS approaches (16S rRNA gene sequencing and whole metagenome sequencing). CONCLUSIONS: Based on our analyses, we consider qPCR with kit-based DNA extraction approaches the best approach to accurately quantify gut bacteria at the strain level in fecal samples. The provided step-by-step protocol will allow scientists to design highly sensitive strain-specific PCR systems for the accurate quantification of bacterial strains of not only L. reuteri but also other bacterial taxa in a broad range of applications and sample types. Video Abstract.

Asunto(s)

Heces , Microbioma Gastrointestinal , Limosilactobacillus reuteri , Humanos , Heces/microbiología , Microbioma Gastrointestinal/genética , Limosilactobacillus reuteri/genética , Limosilactobacillus reuteri/clasificación , Reproducibilidad de los Resultados , ADN Bacteriano/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Límite de Detección , Sensibilidad y Especificidad , Bacterias/genética , Bacterias/clasificación , Bacterias/aislamiento & purificación

RESUMEN

Culturing and genomic sequencing of Mycobacterium tuberculosis (MTB) from tuberculosis (TB) cases is the basis for many research and clinical applications. The alternative, culture-free sequencing from diagnostic samples, is promising but poses challenges to obtain and analyse the MTB genome. Paradoxically, culture is assumed to impose a diversity bottleneck, which, if true, would entail unexplored consequences. To unravel this paradox we generate high-quality genomes of sputum-culture pairs from two different settings after developing a workflow for sequencing from sputum and a tailored bioinformatics analysis. Careful downstream comparisons reveal sources of sputum-culture incongruences due to false positive/negative variation associated with factors like low input MTB DNA or variable genomic depths. After accounting for these factors, contrary to the bottleneck dogma, we identify a 97% variant agreement within sputum-culture pairs, with a high correlation also in the variants' frequency (0.98). The combined analysis from five different settings and more than 100 available samples shows that our results can be extrapolated to different TB epidemic scenarios, demonstrating that for the cases tested culture accurately mirrors clinical samples.

Asunto(s)

Variación Genética , Mycobacterium tuberculosis , Esputo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Esputo/microbiología , Humanos , Tuberculosis/microbiología , Tuberculosis/diagnóstico , Genoma Bacteriano , ADN Bacteriano/genética , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/diagnóstico

RESUMEN

The reclassification of Butyrivibrio crossotus Moore et al. 1976 (Approved Lists 1980) as Eshraghiella crossota gen. nov., comb. nov. is proposed within the family Lachnospiraceae. This reclassification is based on differences revealed through the analysis of 16S rRNA, groEL, recA, and rpoB genes, as well as genome sequences, distinguishing it from other Butyrivibrio species. Comparative analysis showed that B. crossotus exhibited digital DNA-DNA hybridization (dDDH) values of 19.40-27.20% and average nucleotide identities based on blast (ANIb) values of 67.06-67.64% with other Butyrivibrio species. These values are significantly below the species delineation thresholds (dDDH, 70%; ANIb, 95-96%), justifying the proposed reclassification. Additionally, the results of the average amino acid identity (AAI) analysis indicated that this species shares 59.22-60.17% AAI with the other species of the genus Butyrivibrio, which is below the AAI threshold (65%) for a genus boundary. In addition, biochemical and morphological characteristics also support the proposal that this species is different from other species of the genus Butyrivibrio. The type strain is ATCC 29175T (DSM 2876T=T9-40AT).

Asunto(s)

Técnicas de Tipificación Bacteriana , ADN Bacteriano , Hibridación de Ácido Nucleico , Filogenia , ARN Ribosómico 16S , Análisis de Secuencia de ADN , ARN Ribosómico 16S/genética , ADN Bacteriano/genética , Clostridiales/clasificación , Clostridiales/genética , Clostridiales/aislamiento & purificación , Ácidos Grasos , Genes Bacterianos

RESUMEN

Nitrite-oxidizing bacteria (NOB), which perform the second step of aerobic nitrification, play an important role in soil. In the present study, we report a novel isolate from agricultural soil affiliated with the genus Nitrobacter and its physiological characteristics. We sampled the surface soil of a vegetable field and obtained mixed culture A31 using the most probable number (MPN) method with inorganic medium containing 0.75| |mM urea (pH 5.5). The dilution-extinction procedure on culture A31 led to the isolation of a strain that was designated as Nitrobacter sp. A67. The nxrB1 gene sequence of Nitrobacter sp. A67 (302 bp) was classified into Cluster 5, and the highest sequence identity was 96.10% with Nitrobacter sp. BS5/19. The NO2- oxidation activity of Nitrobacter sp. A67 was investigated at various pH. The optimum pH for NO2- oxidation was 5.8-6.4. This result indicates that Nitrobacter sp. A67 is a moderately acidophilic nitrite-oxidizing bacterium.

Asunto(s)

Nitrificación , Nitritos , Nitrobacter , Oxidación-Reducción , Filogenia , ARN Ribosómico 16S , Microbiología del Suelo , Urea , Nitrobacter/metabolismo , Nitrobacter/genética , Nitritos/metabolismo , Urea/metabolismo , Concentración de Iones de Hidrógeno , ARN Ribosómico 16S/genética , ADN Bacteriano/genética , Análisis de Secuencia de ADN

RESUMEN

A Gram-stain-negative, yellow-pigmented, and strictly aerobic bacterium, designated as strain MSW5T, was isolated from seawater of the Yellow Sea in South Korea. The cells were non-motile rods exhibiting oxidase- and catalase-positive activities. Growth was observed at 15-25 °C (optimum, 25 °C) and pH 5.0-9.0 (optimum, pH 7.0-8.0) and in the presence of 1.0-5.0% (w/v) NaCl (optimum, 2.0%). Menaquinone-6 was the sole respiratory quinone, and iso-C15 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), iso-C15 : 0 3-OH, and C15 : 1 ω6c were the major cellular fatty acids. Major polar lipids included phosphatidylethanolamine, two unidentified aminolipids, and three unidentified lipids. Phylogenetic analyses based on 16S rRNA gene sequences and 92 concatenated core protein sequences revealed that strain MSW5T formed a distinct lineage within the genus Polaribacter. The genome of strain MSW5T was 3582 kb in size with a 29.1 mol% G+C content. Strain MSW5T exhibited the highest similarity to Polaribacter atrinae WP25T, with a 97.9% 16S rRNA gene sequence similarity. However, the average nucleotide identity and digital DNA-DNA hybridization values were 79.4 and 23.3%, respectively, indicating that strain MSW5T represents a novel species. Based on its phenotypic, chemotaxonomic, and phylogenetic characteristics, strain MSW5T is proposed to represent a novel species, with the name Polaribacter ponticola sp. nov. The type strain is MSW5T (=KACC 22340T=NBRC 116025T). In addition, whole genome sequence comparisons and phenotypic features suggested that Polaribacter sejongensis and Polaribacter undariae belong to the same species, with P. undariae proposed as a later heterotypic synonym of P. sejongensis. An emended description of Polaribacter sejongensis is also proposed.

Asunto(s)

Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano , Ácidos Grasos , Filogenia , ARN Ribosómico 16S , Agua de Mar , Análisis de Secuencia de ADN , Vitamina K 2 , ARN Ribosómico 16S/genética , Ácidos Grasos/análisis , Agua de Mar/microbiología , República de Corea , ADN Bacteriano/genética , Vitamina K 2/análogos & derivados , Vitamina K 2/análisis , Fosfatidiletanolaminas , Hibridación de Ácido Nucleico , Bacteroidetes/genética , Bacteroidetes/clasificación , Bacteroidetes/aislamiento & purificación , Fosfolípidos/análisis , Fosfolípidos/química

RESUMEN

The gut microbiota of infants in low- to middle-income countries is underrepresented in microbiome research. This study explored the faecal microbiota composition and faecal cytokine profiles in a cohort of infants in a rural province of Cambodia and investigated the impact of sample storage conditions and infant environment on microbiota composition. Faecal samples collected at three time points from 32 infants were analysed for microbiota composition using 16S rRNA amplicon sequencing and concentrations of faecal cytokines. Faecal bacterial isolates were subjected to whole genome sequencing and genomic analysis. We compared the effects of two sample collection methods due to the challenges of faecal sample collection in a rural location. Storage of faecal samples in a DNA preservation solution preserved Bacteroides abundance. Microbiota analysis of preserved samples showed that Bifidobacterium was the most abundant genus with Bifidobacterium longum the most abundant species, with higher abundance in breast-fed infants. Most infants had detectable pathogenic taxa, with Shigella and Klebsiella more abundant in infants with recent diarrhoeal illness. Neither antibiotics nor infant growth were associated with gut microbiota composition. Genomic analysis of isolates showed gene clusters encoding the ability to digest human milk oligosaccharides in B. longum and B. breve isolates. Antibiotic-resistant genes were present in both potentially pathogenic species and in Bifidobacterium. Faecal concentrations of Interlukin-1alpha and vascular endothelial growth factor were higher in breast-fed infants. This study provides insights into an underrepresented population of rural Cambodian infants, showing pathogen exposure and breastfeeding impact gut microbiota composition and faecal immune profiles.

Asunto(s)

Bifidobacterium , Citocinas , Diarrea , Heces , Microbioma Gastrointestinal , ARN Ribosómico 16S , Población Rural , Humanos , Heces/microbiología , Lactante , Cambodia , Citocinas/metabolismo , ARN Ribosómico 16S/genética , Femenino , Masculino , Diarrea/microbiología , Bifidobacterium/genética , Bifidobacterium/aislamiento & purificación , Dieta , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Shigella/genética , Shigella/aislamiento & purificación , Bacteroides/genética , Bacteroides/aislamiento & purificación , Klebsiella/genética , Klebsiella/aislamiento & purificación , Lactancia Materna , ADN Bacteriano/genética , Secuenciación Completa del Genoma , Leche Humana/microbiología , Leche Humana/química

RESUMEN

BACKGROUND: Oligotrophy and hypereutrophy represent the two extremes of lake trophic states, and understanding the distribution of bacterial communities across these contrasting conditions is crucial for advancing aquatic microbial research. Despite the significance of these extreme trophic states, bacterial community characteristics and co-occurrence patterns in such environments have been scarcely interpreted. To bridge this knowledge gap, we collected 60 water samples from Lake Fuxian (oligotrophic) and Lake Xingyun (hypereutrophic) during different hydrological periods. RESULTS: Employing 16S rRNA gene sequencing, our findings revealed distinct community structures and metabolic potentials in bacterial communities of hypereutrophic and oligotrophic lake ecosystems. The hypereutrophic ecosystem exhibited higher bacterial α- and ß-diversity compared to the oligotrophic ecosystem. Actinobacteria dominated the oligotrophic Lake Fuxian, while Cyanobacteria, Proteobacteria, and Bacteroidetes were more prevalent in the hypereutrophic Lake Xingyun. Functions associated with methanol oxidation, methylotrophy, fermentation, aromatic compound degradation, nitrogen/nitrate respiration, and nitrogen/nitrate denitrification were enriched in the oligotrophic lake, underscoring the vital role of bacteria in carbon and nitrogen cycling. In contrast, functions related to ureolysis, human pathogens, animal parasites or symbionts, and phototrophy were enriched in the hypereutrophic lake, highlighting human activity-related disturbances and potential pathogenic risks. Co-occurrence network analysis unveiled a more complex and stable bacterial network in the hypereutrophic lake compared to the oligotrophic lake. CONCLUSION: Our study provides insights into the intricate relationships between trophic states and bacterial community structure, emphasizing significant differences in diversity, community composition, and network characteristics between extreme states of oligotrophy and hypereutrophy. Additionally, it explores the nuanced responses of bacterial communities to environmental conditions in these two contrasting trophic states.

Asunto(s)

Bacterias , Biodiversidad , Lagos , Filogenia , ARN Ribosómico 16S , Lagos/microbiología , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , ARN Ribosómico 16S/genética , ADN Bacteriano/genética , Microbiota/genética , Ecosistema , Microbiología del Agua , China , Nitrógeno/metabolismo , Análisis de Secuencia de ADN

RESUMEN

In nature, the number of genome or chromosome copies within cells (ploidy) can vary between species and environmental conditions, potentially influencing how organisms adapt to changing environments. Although ploidy levels cannot be easily determined by standard genome sequencing, understanding ploidy is crucial for the quantitative interpretation of molecular data. Cyanobacteria are known to contain haploid, oligoploid, and polyploid species. The smallest cyanobacteria, picocyanobacteria (less than 2 µm in diameter), have a widespread distribution ranging from marine to freshwater environments, contributing significantly to global primary production. In this study, we determined the ploidy level of genetically and physiologically diverse brackish picocyanobacteria isolated from the Baltic Sea using a qPCR assay targeting the rbcL gene. The strains contained one to four genome copies per cell. The ploidy level was not linked with phylogeny based on the identity of the 16S rRNA gene. The variation of ploidy among the brackish strains was lower compared to what has been reported for freshwater strains and was more similar to what has been reported for marine strains. The potential ecological advantage of polyploidy among picocyanobacteria has yet to be described. Our study highlights the importance of considering ploidy to interpret the abundance and adaptation of brackish picocyanobacteria.

Asunto(s)

Cianobacterias , Filogenia , Ploidias , ARN Ribosómico 16S , Agua de Mar , Agua de Mar/microbiología , Cianobacterias/genética , Cianobacterias/clasificación , Cianobacterias/aislamiento & purificación , ARN Ribosómico 16S/genética , ADN Bacteriano/genética , Océanos y Mares

RESUMEN

Introduction: Mycobacterium tuberculosis, the causative agent of human tuberculosis, poses a significant threat to global public health and imposes a considerable burden on the economy. However, existing laboratory diagnostic methods for M. tuberculosis are time-consuming and have limited sensitivity levels. Methods: The CRISPR/Cas system, commonly known as the "gene scissors", demonstrates remarkable specificity and efficient signal amplification capabilities. Enzymatic recombinase amplification (ERA) was utilized to rapidly amplify trace DNA fragments at a consistent temperature without relying on thermal cyclers. By integrating of CRISPR/Cas12a with ERA, we successfully developed an ERA-CRISPR/Cas12a detection system that enables rapid identification of M. tuberculosis. Results: The sensitivity of the ERA-CRISPR/Cas12a fluorescence and lateral flow systems was 9 copies/µL and 90 copies/µL, respectively. Simultaneously, the detection system exhibited no cross-reactivity with various of respiratory pathogens and non-tuberculosis mycobacteria, demonstrating a specificity of 100%. The positive concordance rate between the ERA-CRISPR/Cas12a fluorescence system and commercial qPCR was 100% in 60 clinical samples. Meanwhile, the lateral flow system showed a positive concordance rate of 93.8% when compared to commercial qPCR. Both methods demonstrated a negative concordance rate of 100%, and the test results can be obtained in 50 min at the earliest. Discussion: The ERA-CRISPR/Cas12a system offers a rapid, sensitive, and specific method that presents a novel approach to laboratory diagnosis of M. tuberculosis.

Asunto(s)

Sistemas CRISPR-Cas , Mycobacterium tuberculosis , Técnicas de Amplificación de Ácido Nucleico , Sensibilidad y Especificidad , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , Tuberculosis/diagnóstico , Tuberculosis/microbiología , Recombinasas/metabolismo , Recombinasas/genética , Técnicas de Diagnóstico Molecular/métodos , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Proteínas Asociadas a CRISPR/genética , Endodesoxirribonucleasas

RESUMEN

Three bacterial strains, 1AS14IT, 1AS12I and 6AS6, isolated from root nodules of Acacia saligna, were characterized using a polyphasic approach. Phylogenetic analysis based on rrs sequences placed all three strains within the Rhizobium leguminosarum complex. Further phylogeny, based on 1 756 bp sequences of four concatenated housekeeping genes (recA, atpD, glnII and gyrB), revealed their distinction from known rhizobia species of the R. leguminosarum complex (Rlc), forming a distinct clade. The closest related species, identified as Rhizobium laguerreae, with a sequence identity of 96.4% based on concatenated recA-atpD-glnII-gyrB sequences. The type strain, 1AS14IT, showed average nucleotide identity (ANI) values of 94.9, 94.3 and 94.1% and DNA-DNA hybridization values of 56.1, 57.4 and 60.0% with the type strains of closest known species: R. laguerreae, Rhizobium acaciae and 'Rhizobium indicum', respectively. Phylogenomic analyses using 81 up-to-date bacteria core genes and the Type (Strain) Genome Server pipeline further supported the uniqueness of strains 1AS14IT, 1AS12I and 6AS6. The relatedness of the novel strains to NCBI unclassified Rhizobium sp. (396 genomes) and metagenome-derived genomes showed ANI values from 76.7 to 94.8% with a species-level cut-off of 96%, suggesting that strains 1AS14I, 1AS12I and 6AS6 are a distinct lineage. Additionally, differentiation of strains 1AS14IT, 1AS12I and 6AS6 from their closest phylogenetic neighbours was achieved using phenotypic, physiological and fatty acid content analyses. Based on the genomic, phenotypic and biochemical data, we propose the establishment of a novel rhizobial species, Rhizobium aouanii sp. nov., with strain 1AS14IT designated as the type strain (=DSM 113914T=LMG 33206T). This study contributes to the understanding of microbial diversity in nitrogen-fixing symbioses, specifically within Acacia saligna ecosystems in Tunisia.

Asunto(s)

Acacia , Técnicas de Tipificación Bacteriana , ADN Bacteriano , Ácidos Grasos , Hibridación de Ácido Nucleico , Filogenia , ARN Ribosómico 16S , Rhizobium , Nódulos de las Raíces de las Plantas , Análisis de Secuencia de ADN , Rhizobium/genética , Rhizobium/clasificación , Rhizobium/aislamiento & purificación , ADN Bacteriano/genética , Acacia/microbiología , ARN Ribosómico 16S/genética , Ácidos Grasos/análisis , Túnez , Nódulos de las Raíces de las Plantas/microbiología , Genes Esenciales/genética , Genes Bacterianos , Composición de Base , Simbiosis

RESUMEN

Bacterial culturomics is a set of techniques to isolate and identify live bacteria from complex microbial ecosystems. Despite its potential to revolutionize microbiome research, bacterial culturomics has significant challenges when applied to human gut microbiome studies due to its labor-intensive nature. Therefore, we established a streamlined culturomics approach with minimal culture conditions for stool sample preincubation. We evaluated the suitability of non-selective medium candidates for maintaining microbial diversity during a 30-day incubation period based on 16S rRNA gene amplicon analysis. Subsequently, we applied four culture conditions (two preincubation media under an aerobic/anaerobic atmosphere) to isolate gut bacteria on a large scale from eight stool samples of healthy humans. We identified 8141 isolates, classified into 263 bacterial species, including 12 novel species candidates. Our analysis of cultivation efficiency revealed that seven days of aerobic and ten days of anaerobic incubation captured approximately 91% and 95% of the identified species within each condition, respectively, with a synergistic effect confirmed when selected preincubation media were combined. Moreover, our culturomics findings expanded the coverage of gut microbial diversity compared to 16S rRNA gene amplicon sequencing results. In conclusion, this study demonstrated the potential of a streamlined culturomics approach for the efficient isolation of gut bacteria from human stool samples. This approach might pave the way for the broader adoption of culturomics in human gut microbiome studies, ultimately leading to a more comprehensive understanding of this complex microbial ecosystem.

Asunto(s)

Bacterias , Heces , Microbioma Gastrointestinal , ARN Ribosómico 16S , Humanos , Microbioma Gastrointestinal/genética , ARN Ribosómico 16S/genética , Heces/microbiología , Bacterias/genética , Bacterias/clasificación , Bacterias/aislamiento & purificación , ADN Bacteriano/genética

RESUMEN

Three Gram-stain-negative, aerobic, non-motile, chemoheterotrophic, short-rod-shaped bacteria, designated CDY1-MB1T, CDY2-MB3, and BDY3-MB2, were isolated from three marine sediment samples collected in the eastern Pacific Ocean. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains were related to the genus Aequorivita and close to the type strain of Aequorivita vitellina F4716T (with similarities of 98.0-98.1%). Strain CDY1-MB1T can grow at 15-37 °C (optimum 30 °C) and in media with pH 6-9 (optimum, pH 7), and tolerate up to 10% (w/v) NaCl. The predominant cellular fatty acids of strain CDY1-MB1T were iso-C15 : 0 (20.7%) and iso-C17 : 0 3-OH (12.8%); the sole respiratory quinone was menaquinone 6; the major polar lipids were phosphatidylethanolamine, two unidentified aminolipids and two unidentified polar lipids. The digital DNA-DNA hybridization/average nucleotide identity values between strains CDY1-MB1T, CDY2-MB3, and BDY3-MB2 and A. vitellina F4716T were 24.7%/81.6-81.7%, thereby indicating that strain CDY1-MB1T should represent a novel species of the genus Aequorivita. The genomic DNA G+C contents were 37.6 % in all three strains. Genomic analysis showed the presence of genes related to nitrogen and sulphur cycling, as well as metal reduction. The genetic traits of these strains indicate their possible roles in nutrient cycling and detoxification processes, potentially shaping the deep-sea ecosystem's health and resilience. Based upon the consensus of phenotypic and genotypic analyses, strain CDY1-MB1T should be classified as a novel species of the genus Aequorivita, for which the name Aequorivita flava sp. nov. is proposed. The type strain is CDY1-MB1T (=MCCC 1A16935T=KCTC 102223T).

Asunto(s)

Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano , Ácidos Grasos , Sedimentos Geológicos , Hibridación de Ácido Nucleico , Filogenia , ARN Ribosómico 16S , Agua de Mar , Análisis de Secuencia de ADN , Vitamina K 2 , Sedimentos Geológicos/microbiología , ARN Ribosómico 16S/genética , Ácidos Grasos/química , Océano Pacífico , Vitamina K 2/análogos & derivados , Vitamina K 2/análisis , ADN Bacteriano/genética , Agua de Mar/microbiología , Fosfolípidos/análisis , Fosfatidiletanolaminas , Flavobacteriaceae/aislamiento & purificación , Flavobacteriaceae/genética , Flavobacteriaceae/clasificación

RESUMEN

BACKGROUND: Several discoveries about leprosy indicate that Mycobacterium leprae transmission mainly occurs by inhalation, and the nose is a major port of entry and exit. Molecular probes have shown certain potential for the detection and identification of M. leprae in patients. The aim of this study was to identify M. leprae in nasal swab specimens using polymerase chain reaction (PCR)-based assays followed by gene sequencing methods. This observational study examines 64 anterior nasal swab samples taken from pretreatment leprosy patients, on-treatment and completed leprosy treatment in Bulukumba, South Sulawesi, Indonesia. METHODS: samples were analyzed by molecular detection methods according to the standard methods at the Clinical Microbiology Laboratory of Hasanuddin University. Descriptive statistics were utilized to summarize patient demographics and outcomes. RESULTS: This study uses PCR to detect the M. leprae deoxyribonucleic acid (DNA) from nasal swab specimens. Data were collected from 64 patients with a percentage of male patients 51.54%. Based on the age category, the group 45-46 years was the most frequent (39.05%). PCR detection proline-rich antigen gene of a 531 bp DNA fragment from M. leprae, was positive in eight patients, and they were multibacillary. Furthermore, PCR was positive in 5 (31.25%) of 16 new leprosy patients, 2 (8.69%) of 23 on-treatment patients, and 1 (4%) of 25 treatment completed patients. Based on the results of the phylogenetic tree and analysis of 8 positive results detected by M. leprae from leprosy patients, almost all samples have a level of similarity, except for sample Ua7. CONCLUSIONS: M. leprae cannot grow in vitro, so molecular diagnostic tools were used to confirm the disease. This study predominantly of males with the age above 45 years of age being the most common. Eight M. leprae were positive from nasal swab leprosy patients. The sequencing findings provide insight into the genetic diversity of the genus M. leprae, so it is necessary to consider the detection of whole-genome sequence.

Asunto(s)

ADN Bacteriano , Lepra , Mycobacterium leprae , Reacción en Cadena de la Polimerasa , Humanos , Mycobacterium leprae/genética , Mycobacterium leprae/aislamiento & purificación , Mycobacterium leprae/clasificación , Masculino , Femenino , Persona de Mediana Edad , Adulto , Lepra/microbiología , Lepra/diagnóstico , Indonesia , Adulto Joven , ADN Bacteriano/genética , Adolescente , Anciano , Nariz/microbiología , Análisis de Secuencia de ADN , Filogenia , Niño