Multidimensional mass profiles increase confidence in bacterial identification when using low-resolution mass spectrometers.

Field-forward analytical technologies, such as portable mass spectrometry (MS), enable essential capabilities for real-time monitoring and point-of-care diagnostic applications. Significant and recent investments improving the features of miniaturized mass spectrometers enable various new applications outside of small molecule detection. Most notably, the addition of tandem mass spectrometry scans (MS/MS) allows the instrument to isolate and fragment ions and increase the analytical specificity by measuring unique chemical signatures for ions of interest. Notwithstanding these technological advancements, low-cost, portable systems still struggle to confidently identify clinically significant organisms of interest, such as bacteria, viruses, and proteinaceous toxins, due to the limitations in resolving power. To overcome these limitations, we developed a novel multidimensional mass fingerprinting technique that uses tandem mass spectrometry to increase the chemical specificity for low-resolution mass spectral profiles. We demonstrated the method's capabilities for differentiating four different bacteria, including attentuated strains of Yersinia pestis. This approach allowed for the accurate (>92%) identification of each organism at the strain level using de-resolved matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data to mimic the performance characteristics of miniaturized mass spectrometers. This work demonstrates that low-resolution mass spectrometers, equipped with tandem MS acquisition modes, can accurately identify clinically relevant bacteria. These findings support the future application of these technologies for field-forward and point-of-care applications where high-performance mass spectrometers would be cost-prohibitive or otherwise impractical.

A Metagenome-Based Investigation of Gene Relationships for Non-Substrate-Associated Microbial Phosphorus Cycling in the Water Column of Streams and Rivers.

Phosphorus (P) is a nutrient of primary importance in all living systems, and it is especially important in streams and rivers which are sensitive to anthropogenic P inputs and eutrophication. Microbes are accepted as the primary mineralizers and solubilizers of P improving bioavailability for organisms at all trophic levels. Here, we use a genomics approach with metagenome sequencing of 24 temperate streams and rivers representing a total P (TP) gradient to identify relationships between functional genes, functional gene groupings, P, and organisms within the P biogeochemical cycle. Combining information from network analyses, functional groupings, and system P levels, we have constructed a System Relational Overview of Gene Groupings (SROGG) which is a cohesive system level representation of P cycle gene and nutrient relationships. Using SROGG analysis in concert with other statistical approaches, we found that the compositional makeup of P cycle genes is strongly correlated to environmental P whereas absolute abundance of P genes shows no significant correlation to environmental P. We also found orthophosphate (PO43-) to be the dominant factor correlating with system P cycle gene composition with little evidence of a strong organic phosphorous correlation present even in more oligotrophic streams.

Microbial Community Structure and Function Decoupling Across a Phosphorus Gradient in Streams.

Phosphorus (P) is a key biological element with important and unique biogeochemical cycling in natural ecosystems. Anthropogenic phosphorus inputs have been shown to greatly affect natural ecosystems, and this has been shown to be especially true of freshwater systems. While the importance of microbial communities in the P cycle is widely accepted, the role, composition, and relationship to P of these communities in freshwater systems still hold many secrets. Here, we investigated combined bacterial and archaeal communities utilizing 16S ribosomal RNA (rRNA) gene sequencing and computationally predicted functional metagenomes (PFMs) in 25 streams representing a strong P gradient. We discovered that 16S rRNA community structure and PFMs demonstrate a degree of decoupling between structure and function in the system. While we found that total phosphorus (TP) was correlated to the structure and functional capability of bacterial and archaeal communities in the system, turbidity had a stronger, but largely independent, correlation. At TP levels of approximately 55 µg/L, we see sharp differences in the abundance of numerous ecologically important taxa related to vegetation, agriculture, sediment, and other ecosystem inhabitants.

Multiple Cases of Bacterial Sequence Erroneously Incorporated Into Publicly Available Chloroplast Genomes.

Public sequencing databases are invaluable resources to biological researchers, but assessing data veracity as well as the curation and maintenance of such large collections of data can be challenging. Genomes of eukaryotic organelles, such as chloroplasts and other plastids, are particularly susceptible to assembly errors and misrepresentations in these databases due to their close evolutionary relationships with bacteria, which may co-occur within the same environment, as can be the case when sequencing plants. Here, based on sequence similarities with bacterial genomes, we identified several suspicious chloroplast assemblies present in the National Institutes of Health (NIH) Reference Sequence (RefSeq) collection. Investigations into these chloroplast assemblies reveal examples of erroneous integration of bacterial sequences into chloroplast ribosomal RNA (rRNA) loci, often within the rRNA genes, presumably due to the high similarity between plastid and bacterial rRNAs. The bacterial lineages identified within the examined chloroplasts as the most likely source of contamination are either known associates of plants, or co-occur in the same environmental niches as the examined plants. Modifications to the methods used to process untargeted 'raw' shotgun sequencing data from whole genome sequencing efforts, such as the identification and removal of bacterial reads prior to plastome assembly, could eliminate similar errors in the future.

Widespread bacterial diversity within the bacteriome of fungi.

Knowledge of associations between fungal hosts and their bacterial associates has steadily grown in recent years as the number and diversity of examinations have increased, but current knowledge is predominantly limited to a small number of fungal taxa and bacterial partners. Here, we screened for potential bacterial associates in over 700 phylogenetically diverse fungal isolates, representing 366 genera, or a tenfold increase compared with previously examined fungal genera, including isolates from several previously unexplored phyla. Both a 16 S rDNA-based exploration of fungal isolates from four distinct culture collections spanning North America, South America and Europe, and a bioinformatic screen for bacterial-specific sequences within fungal genome sequencing projects, revealed that a surprisingly diverse array of bacterial associates are frequently found in otherwise axenic fungal cultures. We demonstrate that bacterial associations with diverse fungal hosts appear to be the rule, rather than the exception, and deserve increased consideration in microbiome studies and in examinations of microbial interactions.

The Gut Microbiome and Alcoholic Liver Disease: Ethanol Consumption Drives Consistent and Reproducible Alteration in Gut Microbiota in Mice.

Phenotypic health effects, both positive and negative, have been well studied in association with the consumption of alcohol in humans as well as several other mammals including mice. Many studies have also associated these same health effects and phenotypes to specific members of gut microbiome communities. Here we utilized a chronic plus binge ethanol feed model (Gao-binge model) to explore microbiome community changes across three independent experiments performed in mice. We found significant and reproducible differences in microbiome community assemblies between ethanol-treated mice and control mice on the same diet absent of ethanol. We also identified significant differences in gut microbiota occurring temporally with ethanol treatment. Peak shift in communities was observed 4 days after the start of daily alcohol consumption. We quantitatively identified many of the bacterial genera indicative of these ethanol-induced shifts including 20 significant genera when comparing ethanol treatments with controls and 14 significant genera based on temporal investigation. Including overlap of treatment with temporal shifts, we identified 25 specific genera of interest in ethanol treatment microbiome shifts. Shifts coincide with observed presentation of fatty deposits in the liver tissue, i.e., Alcoholic Liver Disease-associated phenotype. The evidence presented herein, derived from three independent experiments, points to the existence of a common, reproducible, and characterizable "mouse ethanol gut microbiome".

A comparison of computationally predicted functional metagenomes and microarray analysis for microbial P cycle genes in a unique basalt-soil forest.

Here we compared microbial results for the same Phosphorus (P) biogeochemical cycle genes from a GeoChip microarray and PICRUSt functional predictions from 16S rRNA data for 20 samples in the four spatially separated Gotjawal forests on Jeju Island in South Korea. The high homogeneity of microbial communities detected at each site allows sites to act as environmental replicates for comparing the two different functional analysis methods. We found that while both methods capture the homogeneity of the system, both differed greatly in the total abundance of genes detected, as well as the diversity of taxa detected. Additionally, we introduce a more comprehensive functional assay that again captures the homogeneity of the system but also captures more extensive community gene and taxonomic information and depth. While both methods have their advantages and limitations, PICRUSt appears better suited to asking questions specifically related to microbial community P as we did here. This comparison of methods makes important distinctions between both the results and the capabilities of each method and can help select the best tool for answering different scientific questions.