Assessment of the non-lactic acid bacteria microbiota in fresh cucumbers and commercially fermented cucumber pickles brined with 6% NaCl.

Limited documentation of the cucumber fermentation microbiome has impeded the understanding of the role of microbes on the quality of finished products. We characterized the microbiome of fresh and fermented cucumber samples using culture dependent and independent techniques, with an emphasis on the non-lactic acid bacteria (non-LAB) population. Insubstantial microbiome variations were observed among fresh cucumber types with Rhizobium (31.04%), Pseudomonas (14.08%), Pantoea (9.25%), Stenotrophomonas (6.83%), and Acinetobacter (6.5%) prevailing. The relative abundance of LAB remained below 0.4% and 4.0% on fresh cucumbers and day 3 of the fermentations brined with 6% sodium chloride, respectively. Fermentation cover brine samples collected on day 1 harbored Pseudomonas, Pantoea, Stenotrophomonas, Acinetobacter, Comamonas, Wautersiella, Microbacterium, Flavobacterium, Ochrobactrum and the Enterobacteriaceae, Citrobacter, Enterobacter and Kluyvera. Plate counts for presumptive Klebsiella and Pseudomonas from fermentation cover brine samples reached 2.80 ±â€¯0.36 and 2.78 ±â€¯0.83 log of CFU/mL, respectively, in 30% and 60% of the nine tanks scrutinized with selective media. Both genera were found in cover brine samples with pH values at 4.04 ±â€¯0.15. We aim at elucidating whether the low relative abundance of non-LAB in commercial cucumber fermentations, in particular Pseudomonas and Enterobacteriaceae, impacts the quality of fermented cucumbers.

An Attenuated Salmonella enterica Serovar Typhimurium Strain and Galacto-Oligosaccharides Accelerate Clearance of Salmonella Infections in Poultry through Modifications to the Gut Microbiome.

Salmonella is estimated to cause one million foodborne illnesses in the United States every year. Salmonella-contaminated poultry products are one of the major sources of salmonellosis. Given the critical role of the gut microbiota in Salmonella transmission, a manipulation of the chicken intestinal microenvironment could prevent animal colonization by the pathogen. In Salmonella, the global regulator gene fnr (fumarate nitrate reduction) regulates anaerobic metabolism and is essential for adapting to the gut environment. This study tested the hypothesis that an attenuated Fnr mutant of Salmonella enterica serovar Typhimurium (attST) or prebiotic galacto-oligosaccharides (GOS) could improve resistance to wild-type Salmonella via modifications to the structure of the chicken gut microbiome. Intestinal samples from a total of 273 animals were collected weekly for 9 weeks to evaluate the impact of attST or prebiotic supplementation on microbial species of the cecum, duodenum, jejunum, and ileum. We next analyzed changes to the gut microbiome induced by challenging the animals with a wild-type Salmonella serovar 4,[5],12:r:- (Nalr) strain and determined the clearance rate of the virulent strain in the treated and control groups. Both GOS and the attenuated Salmonella strain modified the gut microbiome but elicited alterations of different taxonomic groups. The attST produced significant increases of Alistipes and undefined Lactobacillus, while GOS increased Christensenellaceae and Lactobacillus reuteri The microbiome structural changes induced by both treatments resulted in a faster clearance after a Salmonella challenge.IMPORTANCE With an average annual incidence of 13.1 cases/100,000 individuals, salmonellosis has been deemed a nationally notifiable condition in the United States by the Centers for Disease Control and Prevention (CDC). Earlier studies demonstrated that Salmonella is transmitted by a subset of animals (supershedders). The supershedder phenotype can be induced by antibiotics, ascertaining an essential role for the gut microbiota in Salmonella transmission. Consequently, modulation of the gut microbiota and modification of the intestinal microenvironment could assist in preventing animal colonization by the pathogen. Our study demonstrated that a manipulation of the chicken gut microbiota by the administration of an attenuated Salmonella strain or prebiotic galacto-oligosaccharides (GOS) can promote resistance to Salmonella colonization via increases of beneficial microorganisms that translate into a less hospitable gut microenvironment.

A comparison of sequencing platforms and bioinformatics pipelines for compositional analysis of the gut microbiome.

BACKGROUND: Advancements in Next Generation Sequencing (NGS) technologies regarding throughput, read length and accuracy had a major impact on microbiome research by significantly improving 16S rRNA amplicon sequencing. As rapid improvements in sequencing platforms and new data analysis pipelines are introduced, it is essential to evaluate their capabilities in specific applications. The aim of this study was to assess whether the same project-specific biological conclusions regarding microbiome composition could be reached using different sequencing platforms and bioinformatics pipelines. RESULTS: Chicken cecum microbiome was analyzed by 16S rRNA amplicon sequencing using Illumina MiSeq, Ion Torrent PGM, and Roche 454 GS FLX Titanium platforms, with standard and modified protocols for library preparation. We labeled the bioinformatics pipelines included in our analysis QIIME1 and QIIME2 (de novo OTU picking [not to be confused with QIIME version 2 commonly referred to as QIIME2]), QIIME3 and QIIME4 (open reference OTU picking), UPARSE1 and UPARSE2 (each pair differs only in the use of chimera depletion methods), and DADA2 (for Illumina data only). GS FLX+ yielded the longest reads and highest quality scores, while MiSeq generated the largest number of reads after quality filtering. Declines in quality scores were observed starting at bases 150-199 for GS FLX+ and bases 90-99 for MiSeq. Scores were stable for PGM-generated data. Overall microbiome compositional profiles were comparable between platforms; however, average relative abundance of specific taxa varied depending on sequencing platform, library preparation method, and bioinformatics analysis. Specifically, QIIME with de novo OTU picking yielded the highest number of unique species and alpha diversity was reduced with UPARSE and DADA2 compared to QIIME. CONCLUSIONS: The three platforms compared in this study were capable of discriminating samples by treatment, despite differences in diversity and abundance, leading to similar biological conclusions. Our results demonstrate that while there were differences in depth of coverage and phylogenetic diversity, all workflows revealed comparable treatment effects on microbial diversity. To increase reproducibility and reliability and to retain consistency between similar studies, it is important to consider the impact on data quality and relative abundance of taxa when selecting NGS platforms and analysis tools for microbiome studies.

The Supragingival Biofilm in Early Childhood Caries: Clinical and Laboratory Protocols and Bioinformatics Pipelines Supporting Metagenomics, Metatranscriptomics, and Metabolomics Studies of the Oral Microbiome.

Early childhood caries (ECC) is a biofilm-mediated disease. Social, environmental, and behavioral determinants as well as innate susceptibility are major influences on its incidence; however, from a pathogenetic standpoint, the disease is defined and driven by oral dysbiosis. In other words, the disease occurs when the natural equilibrium between the host and its oral microbiome shifts toward states that promote demineralization at the biofilm-tooth surface interface. Thus, a comprehensive understanding of dental caries as a disease requires the characterization of both the composition and the function or metabolic activity of the supragingival biofilm according to well-defined clinical statuses. However, taxonomic and functional information of the supragingival biofilm is rarely available in clinical cohorts, and its collection presents unique challenges among very young children. This paper presents a protocol and pipelines available for the conduct of supragingival biofilm microbiome studies among children in the primary dentition, that has been designed in the context of a large-scale population-based genetic epidemiologic study of ECC. The protocol is being developed for the collection of two supragingival biofilm samples from the maxillary primary dentition, enabling downstream taxonomic (e.g., metagenomics) and functional (e.g., transcriptomics and metabolomics) analyses. The protocol is being implemented in the assembly of a pediatric precision medicine cohort comprising over 6000 participants to date, contributing social, environmental, behavioral, clinical, and biological data informing ECC and other oral health outcomes.

Exposures Related to House Dust Microbiota in a U.S. Farming Population.

BACKGROUND: Environmental factors can influence the house dust microbiota, which may impact health outcomes. Little is known about how farming exposures impact the indoor microbiota. OBJECTIVE: We aimed to identify exposures related to bacterial communities in house dust in a U.S. farming population. METHODS: We used 16S rRNA amplicon sequencing to characterize bacterial communities in vacuumed dust samples from the bedrooms of a subset of 879 households of farmers and farmers' spouses enrolled in the Agricultural Lung Health Study (ALHS), a case-control study of asthma nested within the Agricultural Health Study (AHS) in North Carolina and Iowa. Information on current farming (past 12 mo), including both crop and animal farming, and other potential microbial sources was obtained via questionnaires. We used linear regression to evaluate associations between exposures and bacterial diversity within each sample, analysis of similarity (ANOSIM), and permutational multivariate analysis of variance (PERMANOVA) to identify exposures related to diversity between samples, and analysis of composition of microbiome to examine whether exposures related to diversity were also related to differential abundance of specific operational taxonomic units (OTUs). RESULTS: Current farming was positively associated with bacterial diversity in house dust, with or without adjustment for nonfarm exposures related to diversity, including presence of indoor pets, home condition, and season of dust collection. Many taxa exhibited differential abundance related to farming. Some taxa in the phyla Chloroflexi and Verrucomicrobia were associated [false discovery rate (FDR)<0.05] with farming but not with other nonfarm factors. Many taxa correlated with the concentration of house dust of endotoxin, commonly studied as a general marker of exposure to the farming environment. CONCLUSIONS: In this farming population, house dust microbiota differed by current farming status. Understanding the determinants of the indoor microbiota is the first step toward understanding potential relationships with health outcomes. https://doi.org/10.1289/EHP3145.

The oral bacterial microbiome of occlusal surfaces in children and its association with diet and caries.

Dental caries is the most prevalent disease in humans globally. Efforts to control it have been invigorated by an increasing knowledge of the oral microbiome composition. This study aimed to evaluate the bacterial diversity in occlusal biofilms and its relationship with clinical surface diagnosis and dietary habits. Anamneses were recorded from thirteen 12-year-old children. Biofilm samples collected from occlusal surfaces of 46 permanent second molars were analyzed by 16S rRNA amplicon sequencing combined with the BLASTN-based search algorithm for species identification. The overall mean decayed, missing and filled surfaces modified index [DMFSm Index, including active white spot lesions (AWSL)] value was 8.77±7.47. Biofilm communities were highly polymicrobial collectively, representing 10 bacterial phyla, 25 classes, 29 orders, 58 families, 107 genera, 723 species. Streptococcus sp_Oral_Taxon_065, Corynebacterium matruchotii, Actinomyces viscosus, Actinomyces sp_Oral_Taxon_175, Actinomyces sp_Oral_Taxon_178, Actinomyces sp_Oral_Taxon_877, Prevotella nigrescens, Dialister micraerophilus, Eubacterium_XI G 1 infirmum were more abundant among surfaces with AWSL, and Streptococcus gordonii, Streptococcus sp._Oral_Taxon_058, Enterobacter sp._str._638 Streptococcus australis, Yersinia mollaretii, Enterobacter cloacae, Streptococcus sp._Oral_Taxon_71, Streptococcus sp._Oral_Taxon_F11, Centipeda sp._Oral_Taxon_D18 were more abundant among sound surfaces. Streptococcus mutans was detected on all surfaces in all patients, while Streptococcus sobrinus was detected only in three patients (mean relative abundances 7.1% and 0.6%, respectively). Neither species differentiated healthy from diseased sites. Diets of nine of the subjects were scored as high in fermentable carbohydrates (≧2X/day between meals). A direct association between relative abundances of bacteria and carbohydrate consumption was observed among 18 species. High consumption of fermentable carbohydrates and sound surfaces were associated with a reduction in bacterial diversity. PCoA plots displayed differences in bacterial community profiles between sound and diseased surfaces. Our study showed that, in addition to mutans streptococci, other species may be associated with the initiation of dental caries on occlusal surfaces, and that biofilm diversity of tooth surfaces is influenced by carbohydrate consumption and a surface's health status.

Bacterial Ecology of Fermented Cucumber Rising pH Spoilage as Determined by Nonculture-Based Methods.

Fermented cucumber spoilage (FCS) characterized by rising pH and the appearance of manure- and cheese-like aromas is a challenge of significant economical impact for the pickling industry. Previous culture-based studies identified the yeasts Pichia manshurica and Issatchenkia occidentalis, 4 Gram-positive bacteria, Lactobacillus buchneri, Lactobacillus parrafaraginis, Clostridium sp., and Propionibacterium and 1 Gram-negative genus, Pectinatus, as relevant in various stages of FCS given their ability to metabolize lactic acid. It was the objective of this study to augment the current knowledge of FCS using culture-independent methods to microbiologically characterize commercial spoilage samples. Ion Torrent data and 16S rRNA cloning library analyses of samples collected from commercial fermentation tanks confirmed the presence of L. rapi and L. buchneri and revealed the presence of additional species involved in the development of FCS such as Lactobacillus namurensis, Lactobacillus acetotolerans, Lactobacillus panis, Acetobacter peroxydans, Acetobacter aceti, and Acetobacter pasteurianus at pH below 3.4. The culture-independent analyses also revealed the presence of species of Veillonella and Dialister in spoilage samples with pH above 4.0 and confirmed the presence of Pectinatus spp. during lactic acid degradation at the higher pH. Acetobacter spp. were successfully isolated from commercial samples collected from tanks subjected to air purging by plating on Mannitol Yeast Peptone agar. In contrast, Lactobacillus spp. were primarily identified in samples of FCS collected from tanks not subjected to air purging for more than 4 mo. Thus, it is speculated that oxygen availability may be a determining factor in the initiation of spoilage and the leading microbiota.

A cellular genetics approach identifies gene-drug interactions and pinpoints drug toxicity pathway nodes.

New approaches to toxicity testing have incorporated high-throughput screening across a broad-range of in vitro assays to identify potential key events in response to chemical or drug treatment. To date, these approaches have primarily utilized repurposed drug discovery assays. In this study, we describe an approach that combines in vitro screening with genetic approaches for the experimental identification of genes and pathways involved in chemical or drug toxicity. Primary embryonic fibroblasts isolated from 32 genetically-characterized inbred mouse strains were treated in concentration-response format with 65 compounds, including pharmaceutical drugs, environmental chemicals, and compounds with known modes-of-action. Integrated cellular responses were measured at 24 and 72 h using high-content imaging and included cell loss, membrane permeability, mitochondrial function, and apoptosis. Genetic association analysis of cross-strain differences in the cellular responses resulted in a collection of candidate loci potentially underlying the variable strain response to each chemical. As a demonstration of the approach, one candidate gene involved in rotenone sensitivity, Cybb, was experimentally validated in vitro and in vivo. Pathway analysis on the combined list of candidate loci across all chemicals identified a number of over-connected nodes that may serve as core regulatory points in toxicity pathways.

In vitro and in vivo mouse models for pharmacogenetic studies.

The identification of causative genes underlying biomedically relevant phenotypes, particularly complex multigenic traits, is of vital interest to modern medicine. Using genome-wide association analysis, many studies have successfully identified thousands of loci (called quantitative trait loci or QTL), some of these associating with drug response phenotypes. However, the determination and validation of putative genes has been much more challenging. The actions of drugs, both efficacious and deleterious, are complex phenotypes that are controlled or influenced in part by genetic mechanisms.Investigation for genetic correlates of complex traits and pharmacogenetic traits is often difficult to perform in human studies due to cost, availability of relevant sample population, and limited ability to control for environmental effects. These challenges can be circumvented with the use of mouse models for pharmacogenetic studies. In addition, the mouse can be treated at sub- and supratherapeutic doses and subjected to invasive procedures, which can facilitate measures of drug response phenotypes, making identification of pharmacogenetically relevant genes more feasible. The availability of multiple mouse genetic and phenotypic resources is an additional benefit to using the mouse for pharmacogenetic studies.Here, we describe the contribution of animal models, specifically the mouse, towards the field of pharmacogenetics. In this chapter, we describe different mouse models, including the knockout mouse, recombinant mouse inbred strains, in vitro mouse cell-based assays, as well as novel experimental approaches like the Collaborative Cross recombinant mouse inbred panel, which can be applied to preclinical pharmacogenetics research. These approaches can be used to assess drug response phenotypes that are difficult to model in humans, thereby facilitating drug discovery, development, and application.