Towards genomic medicine: a tailored next-generation sequencing panel for hydroxyurea pharmacogenomics in Tanzania.

BACKGROUND: Pharmacogenomics of hydroxyurea is an important aspect in the management of sickle cell disease (SCD), especially in the era of genomic medicine. Genetic variations in loci associated with HbF induction and drug metabolism are prime targets for hydroxyurea (HU) pharmacogenomics, as these can significantly impact the therapeutic efficacy and safety of HU in SCD patients. METHODS: This study involved designing of a custom panel targeting BCL11A, ARG2, HBB, HBG1, WAC, HBG2, HAO2, MYB, SAR1A, KLF10, CYP2C9, CYP2E1 and NOS1 as potential HU pharmacogenomics targets. These genes were selected based on their known roles in HbF induction and HU metabolism. The panel was designed using the Illumina Design Studio (Illumina, San Diego, CA, USA) and achieved a total coverage of 96% of all genomic targets over a span of 51.6 kilobases (kb). This custom panel was then sequenced using the Illumina MiSeq platform to ensure high coverage and accuracy. RESULTS: We are reporting a successfully designed Illumina (MiSeq) HU pharmacogenomics custom panel encompassing 51.6 kilobases. The designed panel achieved greater than 1000x amplicon coverage which is sufficient for genomic analysis. CONCLUSIONS: This study provides a valuable tool for research in HU pharmacogenomics, especially in Africa where SCD is highly prevalent, and personalized medicine approaches are crucial for improving patient outcomes. The custom-designed Illumina (MiSeq) panel, with its extensive coverage and high sequencing depth, provides a robust platform for studying genetic variations associated with HU response. This panel can contribute to the development of tailored therapeutic strategies, ultimately enhancing the management of SCD through more effective and safer use of hydroxyurea.

Evaluation of the variations in chemical and microbiological properties of the sourdoughs produced with selected lactic acid bacteria strains during fermentation.

This research aimed to analyze variations in chemical properties, microbiological characteristics and generated volatile organic compounds (VOCs) profile during sourdough fermentation. Sourdoughs were collected from different cities in Turkey at two different times and lactic acid bacteria (LAB) in the samples were identified with culture-independent and culture-dependent molecular methods. According to culture-dependent methodology, thirteen LAB species were identified. Lactobacillus spp. were identified as the major group according to MiSeq Illumina analysis. Technological potential of commonly isolated LAB species was evaluated. Due to high frequency of isolation, Fructilactobacillus sanfranciscensis and Lactiplantibacillus plantarum strains were better investigated for their technological traits useful in sourdough production. Experimental sourdoughs were produced with mono- and dual-culture of the selected strains and chemical properties and microbiological characteristics, as well as VOCs profile of the sourdoughs, were subjected to multivariate analysis which showed the relevance of added starter, in terms of acidification and VOCs profile.

Preliminary Investigation of Biogenic Amines in Type I Sourdoughs Produced at Home and Bakery Level.

During a survey for isolating sourdough lactic acid bacteria (LAB), 20 dough samples produced at the bakery level (BL) or home-made (HM) were collected. An enzyme-based colorimetric method revealed a total biogenic amines (BAs) concentration in the range 41.4-251.8 ppm for six (three BL and three HM) sourdoughs characterised by unpleasant odours. Eight BAs generally investigated in foods were identified and quantified from these six samples by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Only one HM sample contained almost all analysed BAs. Tryptamine was exclusively detected in HM sourdoughs (0.71-24.1 ppm). Putrescine, tryptamine, spermidine, and spermine were the only BAs detected in BL sourdoughs. MiSeq Illumina analysis was applied to study the total bacterial community of sourdoughs. LAB accounted from 67.89 to 92.17% of total bacterial diversity, and Levilactobacillus brevis was identified in all six sourdoughs. Leuconostoc, Pediococcus, and Weissella were also dominant. Plate counts detected neither the presence of Pseudomonas nor members of the Enterobacteriaceae family, and LAB levels were, on average, barely 5.89 Log CFU/g for BL, and 7.33 Log CFU/g for HM sourdoughs. Data suggested that the microorganisms mainly imputable of BAs formation in sourdough are members of the LAB community.

A Multivariate Approach to Study the Bacterial Diversity Associated to the Wooden Shelves Used for Aging Traditional Sicilian Cheeses.

The present study was carried to correlate the microbial diversity of the biofilms developed on the wooden boards used for aging traditional Sicilian cheeses with cheese typology. To this end, the microbial diversity of the shelves in contact with the cheeses PDO Pecorino Siciliano, PDO Piacentinu Ennese, and TAP Caciocavallo Palermitano, during ripening, was evaluated by a multivariate statistical approach. The shelf biofilms of this study were previously analyzed for their microbial composition, but no correlation between biodiversity and cheese type was investigated. Canonical discriminant analysis confirmed a cheese typology effect on the microbial loads of the wooden shelves investigated. Regarding the plate count data, the centroids of different cheeses were statistically distant from one another. This analysis also showed a good graphic separation of data regarding bacterial order operational taxonomy units (OTUs). Thus, the microbiological differences imputed to the cheese typologies were not affected by the environmental conditions of the facilities. Furthermore, wooden shelf lactic acid bacteria (LAB) were investigated for their ability to inhibit the main dairy pathogens. Although inhibitors were mainly enterococci, P. pentosaceus WS287 and W. paramesenteroides WS581 showed the highest inhibition activity, indicating their possible application to control the undesired bacteria in situ.

Life in the Wheat Litter: Effects of Future Climate on Microbiome and Function During the Early Phase of Decomposition.

Even though it is widely acknowledged that litter decomposition can be impacted by climate change, the functional roles of microbes involved in the decomposition and their answer to climate change are less understood. This study used a field experimental facility settled in Central Germany to analyze the effects of ambient vs. future climate that is expected in 50-80 years on mass loss and physicochemical parameters of wheat litter in agricultural cropland at the early phase of litter decomposition process. Additionally, the effects of climate change were assessed on microbial richness, community compositions, interactions, and their functions (production of extracellular enzymes), as well as litter physicochemical factors shaping their colonization. The initial physicochemical properties of wheat litter did not change between both climate conditions; however, future climate significantly accelerated litter mass loss as compared with ambient one. Using MiSeq Illumina sequencing, we found that future climate significantly increased fungal richness and altered fungal communities over time, while bacterial communities were more resistant in wheat residues. Changes on fungal richness and/or community composition corresponded to different physicochemical factors of litter under ambient (Ca2+, and pH) and future (C/N, N, P, K+, Ca2+, pH, and moisture) climate conditions. Moreover, highly correlative interactions between richness of bacteria and fungi were detected under future climate. Furthermore, the co-occurrence networks patterns among dominant microorganisms inhabiting wheat residues were strongly distinct between future and ambient climates. Activities of microbial ß-glucosidase and N-acetylglucosaminidase in wheat litter were increased over time. Such increased enzymatic activities were coupled with a significant positive correlation between microbial (both bacteria and fungi) richness and community compositions with these two enzymatic activities only under future climate. Overall, we provide evidence that future climate significantly impacted the early phase of wheat litter decomposition through direct effects on fungal communities and through indirect effects on microbial interactions as well as corresponding enzyme production.

In-Depth Investigation of the Safety of Wooden Shelves Used for Traditional Cheese Ripening.

The main goal of this research was to characterize the bacterial diversity of the wooden boards used for aging traditional Sicilian cheeses and to evaluate whether pathogenic bacteria are associated with these surfaces. Eighteen cheese dairy factories producing three traditional cheese typologies (PDO Pecorino Siciliano, PDO Piacentinu Ennese, and Caciocavallo Palermitano) were selected within the region of Sicily. The wooden shelf surfaces were sampled by a destructive method to detach wood splinters as well as by a nondestructive brushing to collect microbial cells. Scanning electron microscopy showed the presence of almost continuous bacterial formations on the majority of the shelves analyzed. Yeasts and fungal hyphae were also visualized, indicating the complexity of the plank communities. The amplicon library of the 16S rRNA gene V3-V4 region was paired-end sequenced using the Illumina MiSeq system, allowing the identification of 14 phyla, 32 classes, 52 orders, 93 families, and 137 genera. Staphylococcus equorum was identified from all wooden surfaces, with a maximum abundance of 64.75%. Among cheese-surface-ripening bacteria, Brevibacterium and Corynebacterium were detected in almost all samples. Several halophilic (Halomonas, Tetragenococcus halophilus, Chromohalobacter, Salimicrobium, Marinococcus, Salegentibacter, Haererehalobacter, Marinobacter, and Idiomarinaceae) and moderately halophilic (Salinicoccus, Psychrobacter, and Salinisphaera) bacteria were frequently identified. Lactic acid bacteria (LAB) were present at low percentages in the genera Leuconostoc, Lactococcus, Lactobacillus, Pediococcus, and Streptococcus. The levels of viable microorganisms on the wooden shelves ranged between 2.4 and 7.8 log CFU/cm2. In some cases, LAB were counted at very high levels (8.2 log CFU/cm2). Members of the Enterobacteriaceae family were detected in a viable state for only six samples. Coagulase-positive staphylococci, Salmonella spp., and Listeria monocytogenes were not detected. Seventy-five strains belonged to the genera Leuconostoc, Lactococcus, Pediococcus, Enterococcus, Lactobacillus, and Weissella. IMPORTANCE This study provides evidence for the lack of pathogenic bacteria on the wooden shelves used to ripen internal bacterially ripened semihard and hard cheeses produced in Sicily. These three cheeses are not inoculated on their surfaces, and surface ripening is not considered to occur or, at least, does not occur at the same extent as surface-inoculated smear cheeses. Several bacterial groups identified from the wooden shelves are typically associated with smear cheeses, strongly suggesting that PDO Pecorino Siciliano, PDO Piacentinu Ennese, and Caciocavallo Palermitano cheese rind contributes to their final organoleptic profiles.

The Diplodia Tip Blight Pathogen Sphaeropsis sapinea Is the Most Common Fungus in Scots Pines' Mycobiome, Irrespective of Health Status-A Case Study from Germany.

The opportunistic pathogen Sphaeropsis sapinea (≡Diplodia sapinea) is one of the most severe pathogens in Scots pine, causing the disease Diplodia tip blight on coniferous tree species. Disease symptoms become visible when trees are weakened by stress. Sphaeropsis sapinea has an endophytic mode in its lifecycle, making it difficult to detect before disease outbreaks. This study aims to record how S. sapinea accumulates in trees of different health status and, simultaneously, monitor seasonal and age-related fluctuations in the mycobiome. We compared the mycobiome of healthy and diseased Scots pines. Twigs were sampled in June and September 2018, and filamentous fungi were isolated. The mycobiome was analyzed by high-throughput sequencing (HTS) of the ITS2 region. A PERMANOVA analysis confirmed that the mycobiome community composition significantly differed between growth years (p < 0.001) and sampling time (p < 0.001) but not between healthy and diseased trees. Sphaeropsis sapinea was the most common endophyte isolated and the second most common in the HTS data. The fungus was highly abundant in symptomless (healthy) trees, presenting in its endophytic mode. Our results highlight the ability of S. sapinea to accumulate unnoticed as an endophyte in healthy trees before the disease breaks out, representing a sudden threat to Scots pines in the future, especially with increasing drought conditions experienced by pines.

A Grain-Based SARA Challenge Affects the Composition of Epimural and Mucosa-Associated Bacterial Communities throughout the Digestive Tract of Dairy Cows.

The effects of a subacute ruminal acidosis (SARA) challenge on the composition of epimural and mucosa-associated bacterial communities throughout the digestive tract were determined in eight non-lactating Holstein cows. Treatments included feeding a control diet containing 19.6% dry matter (DM) starch and a SARA-challenge diet containing 33.3% DM starch for two days after a 4-day grain step-up. Subsequently, epithelial samples from the rumen and mucosa samples from the duodenum, proximal, middle and distal jejunum, ileum, cecum and colon were collected. Extracted DNA from these samples were analyzed using MiSeq Illumina sequencing of the V4 region of the 16S rRNA gene. Distinct clustering patterns for each diet existed for all sites. The SARA challenge decreased microbial diversity at all sites, with the exception of the middle jejunum. The SARA challenge also affected the relative abundances of several major phyla and genera at all sites but the magnitude of these effects differed among sites. In the rumen and colon, the largest effects were an increase in the relative abundance of Firmicutes and a reduction of Bacteroidetes. In the small intestine, the largest effect was an increase in the relative abundance of Actinobacteria. The grain-based SARA challenge conducted in this study did not only affect the composition and cause dysbiosis of epimural microbiota in the rumen, it also affected the mucosa-associated microbiota in the intestines. To assess the extent of this dysbiosis, its effects on the functionality of these microbiota must be determined in future.

In Vivo Genome and Methylome Adaptation of cag-Negative Helicobacter pylori during Experimental Human Infection.

Multiple studies have demonstrated rapid bacterial genome evolution during chronic infection with Helicobacter pylori In contrast, little was known about genetic changes during the first stages of infection, when selective pressure is likely to be highest. Using single-molecule, real-time (SMRT) and Illumina sequencing technologies, we analyzed genome and methylome evolution during the first 10 weeks of infection by comparing the cag pathogenicity island (cagPAI)-negative H. pylori challenge strain BCS 100 with pairs of H. pylori reisolates from gastric antrum and corpus biopsy specimens of 10 human volunteers who had been infected with this strain as part of a vaccine trial. Most genetic changes detected in the reisolates affected genes with a surface-related role or a predicted function in peptide uptake. Apart from phenotypic changes of the bacterial envelope, a duplication of the catalase gene was observed in one reisolate, which resulted in higher catalase activity and improved survival under oxidative stress conditions. The methylomes also varied in some of the reisolates, mostly by activity switching of phase-variable methyltransferase (MTase) genes. The observed in vivo mutation spectrum was remarkable for a very high proportion of nonsynonymous mutations. Although the data showed substantial within-strain genome diversity in the challenge strain, most antrum and corpus reisolates from the same volunteers were highly similar to each other, indicating that the challenge infection represents a major selective bottleneck shaping the transmitted population. Our findings suggest rapid in vivo selection of H. pylori during early-phase infection providing adaptation to different individuals by common mechanisms of genetic and epigenetic alterations.IMPORTANCE Exceptional genetic diversity and variability are hallmarks of Helicobacter pylori, but the biological role of this plasticity remains incompletely understood. Here, we had the rare opportunity to investigate the molecular evolution during the first weeks of H. pylori infection by comparing the genomes and epigenomes of H. pylori strain BCS 100 used to challenge human volunteers in a vaccine trial with those of bacteria reisolated from the volunteers 10 weeks after the challenge. The data provide molecular insights into the process of establishment of this highly versatile pathogen in 10 different human individual hosts, showing, for example, selection for changes in host-interaction molecules as well as changes in epigenetic methylation patterns. The data provide important clues to the early adaptation of H. pylori to new host niches after transmission, which we believe is vital to understand its success as a chronic pathogen and develop more efficient treatments and vaccines.

Addition of selected starter/non-starter lactic acid bacterial inoculums to stabilise PDO Pecorino Siciliano cheese production.

The present study was carried out to produce Protected Denomination of Origin (PDO) Pecorino Siciliano cheese with a multi-species lactic acid bacteria (LAB) culture, composed of starter and non-starter strains in order to reduce the microbiological variability of the products derived without LAB inoculums. To this end, cheese samples produced in six factories located in five provinces (Agrigento, Catania, Enna, Palermo and Trapani) of Sicily, and previously characterised for physicochemical, microbiological and sensory aspects, have been investigated in this work for bacterial microbiome, fatty acid (FA) composition as well as volatile organic compound (VOC) profiles. Analysis of the cheese microbiomes indicated that streptococci (30.62-77.18% relative abundance) and lactobacilli (on average 25.90% relative abundance) dominated the bacterial communities of control cheeses, produced without exogenous inoculums, whereas the cheeses produced with the selected multi-strain culture saw the dominance of lactococci (in the range 6.49-14.92% relative abundance), streptococci and lactobacilli. After the addition of the selected mixed culture, Shannon index increased in all cheeses, but only the cheeses produced with the selected LAB mixed culture in the factory 2 showed Gini-Simpson diversity index (0.79) closer to the reference value (0.94) for a perfect even community. FA composition, mainly represented by saturated FA (on average 69.60% and 69.39% in control cheeses and experimental cheeses, respectively), was not affected by adding LAB culture. The presence of polyunsaturated FA ranged between 7.93 and 8.03% of FA. VOC profiles were different only for the content of butanoic acid, registered for the experimental cheeses at higher concentrations (on average 662.54 mg/kg) than control cheeses (barely 11.96 mg/kg). This study validated addition of the ad hoc starter/non-starter culture for PDO Pecorino cheese production.

Future Climate Significantly Alters Fungal Plant Pathogen Dynamics during the Early Phase of Wheat Litter Decomposition.

Returning wheat residues to the soil is a common practice in modern agricultural systems and is considered to be a sustainable practice. However, the negative contribution of these residues in the form of "residue-borne pathogens" is recognized. Here, we aimed to investigate the structure and ecological functions of fungal communities colonizing wheat residues during the early phase of decomposition in a conventional farming system. The experiment was conducted under both ambient conditions and a future climate scenario expected in 50-70 years from now. Using MiSeq Illumina sequencing of the fungal internal transcribed spacer 2 (ITS2), we found that plant pathogenic fungi dominated (~87% of the total sequences) within the wheat residue mycobiome. Destructive wheat fungal pathogens such as Fusarium graminearum, Fusarium tricinctum, and Zymoseptoria tritci were detected under ambient and future climates. Moreover, future climate enhanced the appearance of new plant pathogenic fungi in the plant residues. Our results based on the bromodeoxyuridine (BrdU) immunocapture technique demonstrated that almost all detected pathogens are active at the early stage of decomposition under both climate scenarios. In addition, future climate significantly changed both the richness patterns and the community dynamics of the total, plant pathogenic and saprotrophic fungi in wheat residues as compared with the current ambient climate. We conclude that the return of wheat residues can increase the pathogen load, and therefore have negative consequences for wheat production in the future.

Shift in the cow milk microbiota during alpine pasture as analyzed by culture dependent and high-throughput sequencing techniques.

In the present study, two groups of cows from a permanent lowland farm (PF) were divided during summer and reared in the PF or in a temporary alpine farm (ALP), respectively. Microbiological analyses were performed with the objective to investigate the microbial evolution of milk before, during, and after summer transhumance comparing, in particular, the two groups of cows to determine whether the alpine pasture could directly influence the milk microbiota. A significant increase of all microbial groups was registered in milk samples collected in the ALP. Interestingly, many strains belonging to species with well reported technological and probiotic activities were isolated from Alpine milk (20% Lactococcus lactis subsp. lactis/cremoris, 18% Lactobacillus paracasei, 14% Bifidobacterium crudilactis and 18% Propionibacterium sp.), whereas only 16% of strains isolated from the permanent farm milk belonged to the species Lactococcus lactis subsp. lactis/cremoris, 6% to Lactobacillus paracasei, 2% to Bifidobacterium crudilactis and 5% to Propionibacterium sp. The MiSeq Illumina data showed that Alpine milk presented a significant reduction of Pseudomonas and an increase of Lactococcus, Bifidobacterium and Lactobacillus genera. These data confirmed the practice of Alpine pasture as one of the main drivers affecting the milk microbiota. All the microbial changes disappeared when cows were delivered back from Alpine pasture to the indoor farm.

Microbial dynamics in durum wheat kernels during aging.

In the present work the microbial dynamics in wheat kernels were evaluated over time. The main aim of this research was to study the resistance of lactic acid bacteria (LAB) and yeasts associated to unprocessed cereals used for bread making during long term conservation. To this purpose four Triticum durum Desf. genotypes including two modern varieties (Claudio and Simeto) and two Sicilian wheat landraces (Russello and Timilia) were analysed by a combined culture-independent and -dependent microbiological approach after one, two or three years from cultivation and threshing. DNA based MiSeq Illumina technology was applied to reveal the entire bacterial composition of all semolina samples. The samples showed a different distribution of bacterial taxa per variety and time of storage. The groups mostly represented were Stenotrophomonas, Pseudomonas, Erwinia, Delftia and Sphingomonas genera, Enterobacteriaceae and Oxalobacteriaceae families, and Actinobacteria phylum. Among LAB, only Enterococcus genus was detected barely in a single sample (Simeto stored for one year) by the next generation sequencing, indicating that LAB remained unassigned or their abundances were below 0.1% or their DNAs were rendered inaccessible. Plate counts showed consistent differences in relation to genotypes and duration of storage, with the highest levels found for total mesophilic microorganisms detected up 6.8 Log CFU/g. Colonies of presumptive sourdough LAB were detected only in a few samples. Cocci constituted the major group of LAB in almost all samples. Following the enrichment procedure, almost all samples were characterised by the presence of acidifying microorganisms. All isolates collected before and after enrichment represented 28 different strains belonging to 10 species of Enterococcus, Lactobacillus and Pediococcus genera. The most resistant species during aging were Enterococcus faecium, Enterococcus durans, Lactobacillus brevis, Lactobacillus pentosus and Lactobacillus paracasei demonstrating that lactobacilli and enterococci are able to overcome the stressing conditions represented by cereal storage better than other LAB genera commonly found associated to cereals after harvest. Yeast community included mostly species with no interest in bread making.

Evolution of indigenous starter microorganisms and physicochemical parameters in spontaneously fermented beef, horse, wild boar and pork salamis produced under controlled conditions.

The present work was carried out to evaluate the microbiological and physicochemical composition of salamis produced with the meat of beef, horse, wild boar and pork. Salami productions occurred under controlled laboratory conditions to exclude butchery environmental contaminations, without the addition of nitrate and nitrite. All trials were monitored during the ripening (13 °C and 90% relative humidity) extended until 45 d. The evolution of physicochemical parameters showed that beef and pork salamis were characterized by a higher content of branched chain fatty acids (FA) and rumenic acid than horse and wild boar salamis, whereas the last two productions showed higher values of secondary lipid oxidation. Plate counts showed that lactic acid bacteria (LAB), yeasts and coagulase-negative staphylococci (CNS) populations dominated the microbial community of all productions with Lactobacillus and Staphylococcus as most frequently isolated bacteria. The microbial diversity evaluated by MiSeq Illumina showed the presence of members of Gammaproteobacteria phylum, Moraxellaceae family, Acinetobacter, Pseudomonas, Carnobacterium and Enterococcus in all salamis. This study showed the natural evolution of indigenous fermented meat starter cultures and confirmed a higher suitability of horse and beef meat for nitrate/nitrite free salami production due to their hygienic quality at 30 d.

Acinetobacter sp. as the key player in diesel oil degrading community exposed to PAHs and heavy metals.

The aim of this study was to verify the hypothesis that a hydrocarbon degrading community isolated from a site heavily polluted with polycyclic aromatic hydrocarbons (PAHs) and heavy metals should exhibit a high activity and biodegradation efficiency, despite decreased biodiversity resulting from the presence of such contaminants. Microbial community isolated from soil collected at an abandoned creosote railway wood-sleepers impregnation plant using diesel oil was used during the studies. Four parallel systems spiked with diesel oil, diesel oil + PAHs, diesel oil + heavy metals and diesel oil + PAHs + heavy metals were analysed in terms of relative abundance and biodiversity of the microbial community (Illumina), biodegradation efficiency (GCMS) and cellular metabolic activity (flow cytometry). Principal Component Analysis and biodiversity parameters indicated that the mixture of PAHs and heavy metals was the dominant factor which resulted in the enrichment of the Gammaproteobacteria class. This was associated with higher degradation of additional PAHs in the presence of heavy metals and an increase of metabolically active sub-populations during flow cytometry analysis. The increased abundance of the Acinetobacter genus in systems with both PAHs and heavy metals implies that it may play a crucial role in soil populations exposed to mixed contaminations.

Breeding farm, level of feeding and presence of antibiotics in the feed influence rabbit cecal microbiota.

BACKGROUND: The effect of the production environment and different management practices in rabbit cecal microbiota remains poorly understood. While previous studies have proved the impact of the age or the feed composition, research in the breeding farm and other animal management aspects, such as the presence of antibiotics in the feed or the level of feeding, is still needed. Characterization of microbial diversity and composition of growing rabbits raised under different conditions could help better understand the role these practices play in cecal microbial communities and how it may result in different animal performance. RESULTS: Four hundred twenty-five meat rabbits raised in two different facilities, fed under two feeding regimes (ad libitum or restricted) with feed supplemented or free of antibiotics, were selected for this study. A 16S rRNA gene-based assessment through the MiSeq Illumina sequencing platform was performed on cecal samples collected from these individuals at slaughter. Different univariate and multivariate approaches were conducted to unravel the influence of the different factors on microbial alpha diversity and composition at phylum, genus and OTU taxonomic levels. The animals raised in the facility harboring the most stable environmental conditions had greater, and less variable, microbial richness and diversity. Bootstrap univariate analyses of variance and sparse partial least squares-discriminant analyses endorsed that farm conditions exerted an important influence on rabbit microbiota since the relative abundances of many taxa were found differentially represented between both facilities at all taxonomic levels characterized. Furthermore, only five OTUs were needed to achieve a perfect classification of samples according to the facility where animals were raised. The level of feeding and the presence of antibiotics did not modify the global alpha diversity but had an impact on some bacteria relative abundances, albeit in a small number of taxa compared with farm, which is consistent with the lower sample classification power according to these factors achieved using microbial information. CONCLUSIONS: This study reveals that factors associated with the farm effect and other management factors, such as the presence of antibiotics in the diet or the feeding level, modify cecal microbial communities. It highlights the importance of offering a controlled breeding environment that reduces differences in microbial cecal composition that could be responsible for different animal performance.

groEL Gene-Based Phylogenetic Analysis of Lactobacillus Species by High-Throughput Sequencing.

Lactobacillus is a fairly diverse genus of bacteria with more than 260 species and subspecies. Many profiling methods have been developed to carry out phylogenetic analysis of this complex and diverse genus, but limitations remain since there is still a lack of comprehensive and accurate analytical method to profile this genus at species level. To overcome these limitations, a Lactobacillus-specific primer set was developed targeting a hypervariable region in the groEL gene-a single-copy gene that has undergone rapid mutation and evolution. The results showed that this methodology could accurately perform taxonomic identification of Lactobacillus down to the species level. Its detection limit was as low as 104 colony-forming units (cfu)/mL for Lactobacillus species. The assessment of detection specificity using the LactobacillusgroEL profiling method found that Lactobacillus, Pediococcus, Weissella, and Leuconostoc genus could be distinguished, but non-Lactobacillus Genus Complex could not be detected. The groEL gene sequencing and Miseq high-throughput approach were adopted to estimate the richness and diversity of Lactobacillus species in different ecosystems. The method was tested using kurut (fermented yak milk) samples and fecal samples of human, rat, and mouse. The results indicated that Lactobacillus mucosae was the predominant gut Lactobacillus species among Chinese, and L. johnsonii accounted for the majority of lactobacilli in rat and mouse gut. Meanwhile, L. delbrueckii subsp. bulgaricus had the highest relative abundance of Lactobacillus in kurut. Thus, this groEL gene profiling method is expected to promote the application of Lactobacillus for industrial production and therapeutic purpose.

Microbial community structure in the rhizosphere of the orphan legume Kersting's groundnut [Macrotyloma geocarpum (Harms) Marechal & Baudet].

The presence of microbial communities in the rhizosphere of plants is an important determinant of plant health and soil organic matter composition. Plant species play significant roles in selecting the specific microbial communities that inhabit the root zone. However, till now, there is no solid information regarding the presence of specific plant-microbiome in the rhizosphere of many plants, especially under-exploited and under-researched species such as Kersting's groundnut. This study assessed the effect of five Kersting's groundnut landraces on the structure of microbial communities in rhizosphere of field-grown plants. The five tested Kersting's groundnut landraces (Belane Mottled, Boli, Funsi, Puffeun and Heng Red Mottled) were found to exert a marked selective influence on bacteria associated with their rhizospheres, measured using 16S rDNA MiSeq illumina sequencing. Community differences in microbial composition and relative abundance were both significant. Numerous phyla in the rhizosphere were affected by the test landraces. Except for Belane mottled whose rhizospheres were dominated by Proteobacteria, the rhizosphere soils of the other landraces were dominated by Bacteroidetes. With the exception of landrace Puffeun which showed only Mesorhizobium in its rhizosphere, all the other test landraces revealed the presence of Bradyrhizobium and Rhizobium species of alpha Proteobacteria. Furthermore, the rhizosphere of all landraces were abundant in species of the indole-3-acetic-acid producing Sphingomonas and cellulose-degrading Fibrobacteres. The results of this study suggest that Kersting's groundnut landraces can shape bacterial community composition in the rhizosphere via plant-related changes in the rhizosphere soil.

Production of Naturally γ-Aminobutyric Acid-Enriched Cheese Using the Dairy Strains Streptococcus thermophilus 84C and Lactobacillus brevis DSM 32386.

The cheese-derived strains Streptococcus thermophilus 84C isolated from Nostrano cheese, and Lactobacillus brevis DSM 32386 isolated from Traditional Mountain Malga cheese have been previously reported as γ-aminobutyric acid (GABA)-producers in vitro. In the present study, the ability of these strains to produce GABA was studied in experimental raw milk cheeses, with the aim to investigate the effect of the culture and the ripening time on the GABA concentration. The cultures used consisted on S. thermophilus 84C alone (84C) or in combination with L. brevis DSM 32386 (84C-DSM). The control culture was a commercial S. thermophilus strain, which was tested alone (CTRL) or in combination with the L. brevis DSM 32386 (CTRL-DSM). The pH evolution, microbiological counts, MiSeq Illumina and UHPLC-HQOMS analysis on milk and cheese samples were performed after 2, 9, and 20 days ripening. During the whole ripening, the pH was always under 5.5 in all batches. The concentration of GABA increased during ripening, with the highest content in 84C after 9 days ripening (84 ± 37 mg/kg), in 84C-DSM and CTRL-DSM after 20 days ripening (91 ± 28 and 88 ± 24 mg/kg, respectively). The data obtained support the hypothesis that S. thermophilus 84C and L. brevis DSM 32386 could be exploited as functional cultures, improving the in situ bio-synthesis of GABA during cheese ripening.

Negligible seeding source effect on the final ANAMMOX community under steady and high nitrogen loading rate after enrichment using poly(vinyl alcohol) gel carriers.

This study investigated the effect of seeding source on the mature anaerobic ammonia oxidation (ANAMMOX) bacterial community niche in continuous poly(vinyl alcohol) (PVA) gel systems operated under high nitrogen loading rate (NLR) condition. Four identical column reactors packed with PVA gels were operated for 182 d using different seeding sources which had distinct community structures. The ANAMMOX reaction was achieved in all the bioreactors with comparable total and ANAMMOX bacterial 16S rRNA gene quantities. The bacterial community structure of the bioreactors became similar during operation; some major bacteria were commonly found. Interestingly, one ANAMMOX species, "Candidatus Brocadia sinica", was conclusively predominant in all the bioreactors, even though different seeding sludges were used as inoculum source, possibly due to the unique physiological characteristics of "Ca. Brocadia sinica" and the operating conditions (i.e., PVA gel-based continuous system and 1.0 kg-N/(m3·d) of NLR). The results clearly suggest that high NLR condition is a more significant factor determining the final ANAMMOX community niche than is the type of seeding source.