This study aimed to examine and compare the effectiveness of a novel spray-dried inoculant and a commercial freeze-dried additive on the fermentation quality, aerobic stability and bacterial population of vetch-oat silage. An entirely random design used a 3 × 4 factorial arrangement of treatments, with and without lactic acid bacteria (LAB) inoculants and four fermentation periods. Physicochemical parameters, microbiological counts and 16S rRNA gene sequencing analysis on Nanopore MinION were conducted to characterize the ensiling process. Both LAB inoculants increased dry matter, crude protein, lactic, acetic and propionic acid contents, while reducing pH, neutral detergent fibre, ammonia nitrogen/total nitrogen and ethanol concentrations compared to the control group. Overall, the native inoculant decreased the cell load of coliforms, yeasts and moulds. In addition, bio-inoculants enhanced the aerobic stability of vetch-oat intercrops. After ensiling, bacterial alpha diversity decreased noticeably; inoculation reduced the number of observed operational taxonomic units and the Shannon and Simpson indices. Notably, the relative abundance of Lactobacillus in the control group was lower than in treated silages, while the relative values of Staphylococcus increased sharply in the uninoculated group. In conclusion, the native strains showed promise for usage as a bio-inoculant in the ensiling of vetch-oat at a mixture rate of 1:1, producing an immediate impact as well as a favourable effect on the post-opening phase. This represents the first report on 16S rRNA gene-based nanopore metagenomics applied to the bacterial analysis of vetch-oat silage, providing a microbiological insight where native and commercial strains dominate the natural epiphytic community.

The genus Lysmata Risso, 1816 includes five species in the Mediterranean Sea, namely two well-known species with an Atlantic–Mediterranean distribution, L. seticaudata (Risso, 1816) and L. nilita Dohrn & Holthuis, 1950, and three additional species only known from single records. These are L. kempi Chace, 1997 and L. vittata (Stimpson, 1860), alien species native to the Indo-Pacific region, and L. olavoi Fransen, 1991, a poorly known species originally described from the Azores (Atlantic Ocean) and only reported three decades ago from the eastern parts of the basin. Based on an integrative taxonomic approach, we report the first finding of an ovigerous female of L. olavoi from Italy (Gulf of Naples, Tyrrhenian Sea) and the entire western Mediterranean, thus confirming the presence of this species in the basin and filling a wide gap in its published distribution. It is possible that this taxon was present in the Mediterranean for centuries but went overlooked or undetected due to cryptic habits, occupancy of still unexplored habitats, or misidentification with the highly similar L. seticaudata. Results of phylogenetic analyses confirm the exclusion of L. olavoi by the major lysmatid groups and underline the necessity of multilocus approaches to disentangle its correct phylogenetic position. As the species is still surrounded by a number of unsolved ecological and phylogenetic questions, further field and laboratory work is required to shed light on them.

The evaluation of symbiotic microbial communities occurring in the intestinal tract of animals has received great interest in recent years. However, little is known about gut microbial communities in cetaceans, despite their relevance in the ecology of marine communities. Here, we report an investigation using 16S rRNA gene amplicon sequencing of the resident gut microbiota of the two cetacean species Stenella coeruleoalba and Tursiops truncatus by sampling intestinal mucosa from specimens retrieved stranded along the Tyrrhenian coast of Tuscany (Italy). We found an abundance of members from Clostridiaceae and Fusobacteriaceae, which in total accounted for more than 50% of reads, in agreement with gut microbiota composition of other carnivorous mammals. Probably due to the limited number of samples available, sex, preservation status and also species, did not correlate with overall differences in the microbiota. Indeed, a high similarity of the taxonomic (family-level) composition between the gut microbiota of the two species was found. However, Pedobacter spp. was found abundant in amplicon sequencing libraries from S. coeruleoalba, while clostridia were more abundant from T. truncatus samples. Our results shed some light on the gut microbiota composition of two dolphin (S. coeruleoalba and T. truncatus) species, with specimens collected in the wild. Studies with a larger number of individuals are now needed to confirm these first results and evaluate the interspecific differences in relation to sex and age.

A collection of 45 isolates was created based on bacteria isolated from maize, broad bean, wheat, rye and wild plants such as horsetail and burdock. The aim of the current study was to isolate the bacteria, and then identify and assess the degree of genomic diversity. The molecular identification of microsymbionts isolated from the endosphere (root and stem) of plants grown in agricultural soils was performed using 16S rRNA gene sequencing. To evaluate the genomic diversity between strains that occurred in multiple host plants, 18 bacterial isolates representing four species were subjected to denaturing gradient gel electrophoresis. The 16S rDNA analysis assigned all bacterial isolates to ten genera, from which Rhizobium was represented by 19 isolates, Delftia by 11, Agrobacterium by five, Stenotrophomonas by three, Brevundimonas by two and Novosphingobium, Variovorax, Collimonas, Achromobacter and Comamonas by only one isolate. Furthermore, the genomic diversity of the 11 isolates of Delftia sp. was assessed using the BOX – polymerase chain reaction (BOX-PCR) and enterobacterial repetitive intergenic consensus – PCR (ERIC-PCR) methods. Typing patterns and analysis using BOX-PCR and ERIC-PCR data demonstrated similarities among the tested isolates. In general, the results obtained with BOX-PCR and ERIC-PCR were in good agreement. However, a greater degree of differentiation patterns of the genomic DNA was obtained in the ERIC-PCR method.

Bats (Order: Chiroptera) have been widely studied as reservoir hosts for viruses of concern for human and animal health. However, whether bats are equally competent hosts of non-viral pathogens such as bacteria remains an important open question. Here, we surveyed blood and saliva samples of vampire bats from Peru and Belize for hemotropic Mycoplasma spp. (hemoplasmas), bacteria that can cause inapparent infection or anemia in hosts. 16S rRNA gene amplification of blood showed 67% (150/223) of common vampire bats (Desmodus rotundus) were infected by hemoplasmas. Sequencing of the 16S rRNA gene amplicons revealed three novel genotypes that were phylogenetically related but not identical to hemoplasmas described from other (non-vampire) bat species, rodents, humans, and non-human primates. Hemoplasma prevalence in vampire bats was highest in non-reproductive and young individuals, did not differ by country, and was relatively stable over time (i.e., endemic). Metagenomics from pooled D. rotundus saliva from Peru detected non-hemotropic Mycoplasma species and hemoplasma genotypes phylogenetically similar to those identified in blood, providing indirect evidence for potential direct transmission of hemoplasmas through biting or social contacts. This study demonstrates vampire bats host several novel hemoplasmas and sheds light on risk factors for infection and basic transmission routes. Given the high frequency of direct contacts that arise when vampire bats feed on humans, domestic animals, and wildlife, the potential of these bacteria to be transmitted between species should be investigated in future work.

Polymannuronic acid (PM), one of numerous alginates isolated from brown seaweeds, is known to possess antioxidant activities. In this study, we examined its potential role in reducing body weight gain and attenuating inflammation induced by a high-fat and high-sucrose diet (HFD) as well as its effect on modulating the gut microbiome in mice. A 30-d PM treatment significantly reduced the diet-induced body weight gain and blood TAG levels (P<0·05) and improved glucose tolerance in male C57BL/6J mice. PM decreased lipopolysaccharides in blood and ameliorated local inflammation in the colon and the epididymal adipose tissue. Compared with low-fat and low-sucrose diet (LFD), HFD significantly reduced the mean number of species-level operational taxonomic units (OTU) per sample as well as species richness (P<0·05) but did not appear to affect other microbial diversity indices. Moreover, compared with LFD, HFD altered the abundance of approximately 23 % of the OTU detected (log10 linear discriminant analysis (LDA) score>2·0). PM also had a profound impact on the microbial composition in the gut microbiome and resulted in a distinct microbiome structure. For example, PM significantly increased the abundance of a probiotic bacterium, Lactobacillus reuteri (log10 LDA score>2·0). Together, our results suggest that PM may exert its immunoregulatory effects by enhancing proliferation of several species with probiotic activities while repressing the abundance of the microbial taxa that harbor potential pathogens. Our findings should facilitate mechanistic studies on PM as a potential bioactive compound to alleviate obesity and the metabolic syndrome.

Anaplasmosis is caused by a group of obligate intracellular bacteria in the genus Anaplasma, which are transmitted by ticks and infect humans, domestic animals and wildlife. This study was conducted to determine the prevalence and molecular characterization of Anaplasma spp. in semi-wild white yaks sampled in Tianzhu Tibetan Autonomous County, northwest China. Out of 332 samples tested, 35 (10·9%) were positive for Anaplasma spp. The positive rates were 6·2% (20/322) and 5·3% (17/322) for Anaplasma bovis and Anaplasma phagocytophilum in white yaks, respectively. None of the sample was positive for Anaplasma marginale. Two (0·6%) samples were simultaneously positive to A. bovis and A. phagocytophilum. Sequence analysis of the 16S rRNA gene revealed two genotypes (ApG1 and ApG2) of A. phagocytophilum and two sequence types (ST1 and ST2) of A. bovis in white yaks. This study is the first to document the presence of Anaplasma in white yaks. Our findings extend the host range for Anaplasma species and provide more valuable information for the control and management of anaplasmosis in white yaks.

Acquisition of the gastrointestinal microbiota at birth may have long-term health impacts. We longitudinally characterised major microbial communities in the faeces of a cohort of infants using molecular methods. Faecal samples were prospectively obtained at several time points after birth from eighty-three infants. Real-time PCR using SYBR green and primers targeted at 16S rRNA gene sequences were used to quantify Bifidobacterium, Lactobacillus acidophilus group, Bacteroides–Prevotella group, Enterobacteriaceae, Enterococcus, Clostridium coccoides–Eubacterium rectale group, Clostridium leptum group and Staphylococcus. Microbial community abundance was expressed relative to amplification of sequences conserved universally for domain bacteria. Faecal copy number of 16S rRNA genes increased non-significantly from a mean of 4·1 × 109/g on day 1 to 1·1 × 1010/g on day 4. All microbial communities were detected from day 1 after birth. Enterobacteriaceae and lactobacilli predominated on day 1, while bifidobacteria and staphylocci increased on day 4. Bacteroides–Prevotella and C. coccoides–E. rectale increased by day 180. C. leptum was detected in half of the cohort at birth and in a slightly larger percentage by 6 months. Caesarean section was associated with delayed colonisation by several bacterial communities. Higher socio-economic status was associated with more abundant lactobacilli and Bacteroides–Prevotella at 90 and 180 d. Supplemental feeding was associated with a reduction in Enterobacteriaceae. Microbial colonisation of the gut was well established on the first day of birth, and relative abundance of microbial communities was influenced by mode of delivery, socio-economic status and supplemental feeding. These findings may have relevance to infant nutrition and growth.

Bacterial community composition during summer was analysed in surface and bottom waters of the oligotrophic shallow (4.5 m) Lake Limnopolar (Livingston Island, South Shetland Islands, Antarctica), using 16S rRNA gene clone libraries and sequencing. Up to 61% of the 16S rDNA sequences found were closely related to sequences retrieved from lakes, glaciers or polar systems. The distribution of these sequences was not homogeneous, with vertical differences found in both bacterial taxa composition and isolation source of the closest match from GenBank. In the surface sample 86% of the sequences were related to bacteria found in soils, seawater or gut microbiota, probably explained by waterborne transport from the catchment, by wind through sea sprays, or local bird activity. Conversely, in the deep samples, 95% of the sequences were closer to bacteria typically described for lakes, glaciers or polar systems. The presence of benthic mosses covering the bottom of the lake favours a more stable deep layer leading to the existence of this biological heterogeneity through the water column, although the lake does not show physical-chemical stratification in summer. This study illustrates a strong influence of external factors on the microbial ecology of this model Antarctic lake.

Although the lower size limit of microorganisms was previously believed to be c. 0.2 μm, there is evidence for the existence of microorganisms that can pass through 0.2 μm-pore-size filters called ultramicrobacteria or nanobacteria. However, information on the phylogeny and biogeography of these bacteria is limited. We obtained 53 isolates of 0.2 μm-passable bacteria from 31 samples collected at 26 locations worldwide, including the Arctic Svalbard Islands, deserts, and Maritime Antarctica. Phylogenetic analysis of near full-length 16S rRNA gene sequences revealed that 18 of the 53 isolates were < 97% homologous with previously cultured isolates, representing potentially novel species. Two isolates (order Rhizobiales) (100% identical) collected from Byers Peninsula, Livingston Island in Maritime Antarctica, were closely related (99.8% similarity) to an isolate collected from intertidal sediments in East Antarctica. In addition, the sequence of this Antarctic isolate showed ≥ 97% similarity to 901 sequences derived from known isolates and samples collected at geographically disparate locations under various environmental conditions. Interestingly, among 13 sequences showing ≥ 99% similarity, ten were isolated from cryospheric habitats such as Arctic, Antarctic, and alpine environments. This implies that such Rhizobiales strains occur in the cryospheric regions, however, their abundance and biomass may be scarce depending on the geographic location.

The modern molecular biology movement was developed in the 1960s with the conglomeration of biology, chemistry, and physics. Today, molecular biology is an integral part of studies aimed at understanding the evolution and ecology of gastrointestinal microbial communities. Molecular techniques have led to significant gains in our understanding of the chicken gastrointestinal microbiome. New advances, primarily in DNA sequencing technologies, have equipped researchers with the ability to explore these communities at an unprecedented level. A reinvigorated movement in systems biology offers a renewed promise in obtaining a more complete understanding of chicken gastrointestinal microbiome dynamics and their contributions to increasing productivity, food value, security, and safety as well as reducing the public health impact of raising production animals. Here, we contextualize the contributions molecular biology has already made to our understanding of the chicken gastrointestinal microbiome and propose targeted research directions that could further exploit molecular technologies to improve the economy of the poultry industry.

Yoghurt and starter culture producers are still searching strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to produce healthier yogurt with longer shelf life, better texture, taste and quality. However, selective identification of Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus from a mixed population using microbiological and biochemical methods is difficult, time consuming and may not be accurate. In this study, a quick, sensitive and accurate method is proposed to identify both Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus using PCR. The method is comprised of two parts. In the first part, methionine biosynthesis genes, known to be present in both species were partially amplified by designed primers (cysmet2F and cysmet2R). Partial amplification of the methionine biosynthesis gene which gives 700 bp fragment resulted in selective identification of Lb. bulgaricus and Strep. thermophilus. All 16 Lb. bulgaricus and 6 Strep. thermophilus isolates assessed by this method gave the expected amplification. On the other hand, further analysis of other closely related species with the same primers have indicated that the same product was also amplified in two more lactobacilli namely, Lb. delbrueckii subsp. lactis and Lb. helveticus species. Thus, in the second part of the method, further differentiation of Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus from each other and these species was achieved using restriction analysis of 16S rRNA gene with EcoRI.

Seven independent clone libraries were constructed to study the biodiversity of the bacterioplankton in the surface waters around Southern Thule, South Sandwich Islands, in order to identify the species present, to determine the sample effort required to estimate the total diversity, and to determine whether the surface waters around Southern Thule represented a highly specialized local anomaly or a subset of the marine meta-community. In total, 672 clones generated 629 useable sequences. These 629 clones matched 278 different sequences deposited in the 16S rDNA sequence databases. The majority of the clones were related to marine microorganisms, many of which had been previously detected in permanently cold Arctic and Antarctic marine environments. Each clone library generated an average of 35.8 new sequence matches. 346 clones covered two-thirds of the total estimated diversity, while 438 clones covered three-quarters of the total estimated diversity. Above this number, the coverage tended to stabilize and a relatively large number of additional clones were required to improve coverage significantly, increasing at the rate of about one new sequence match per 100 new clones. Comparing the different clone libraries, eight matches occurred in each of the seven libraries, whilst fifty-five occurred in only one, suggesting that there might be a relatively small number of common dominant ubiquitous species, with a much larger underlying diversity or ‘seed bank’ from which this dominant diversity is drawn. This study suggests that the dominant bacterioplankton in the surface waters around Southern Thule represent a subset of the marine meta-community, whilst sub-dominant diversity appears to be a highly specialized local anomaly.

As a result of the recent application of DNA based technology to the investigation of maritime Antarctic freshwater lakes, patterns have begun to emerge in the bacterioplankton communities that dominate these systems. In this study, the bacterioplankton communities of five Antarctic and five Arctic freshwater lakes were assessed and compared with existing data in the literature, to determine whether emerging patterns in Antarctic lakes also applied to Arctic systems. Such a bipolar comparison is particularly timely, given the current interest in biogeography, the global distribution of microorganisms and the controversy over the global ubiquity hypothesis. In addition, it has recently been discovered that commonly encountered bacterial sequences, often originating from uncultivated bacteria obtained on different continents, form coherent phylogenetic freshwater clusters. In this study we encountered both identical sequences and sequences with a high degree of similarity among the bacterioplankton in lake water from both poles. In addition, Arctic freshwater lakes appeared to be dominated by some of the same groups of bacterioplankton thought to be dominant in Antarctic lakes, the vast majority of which represented uncultivated groups.

Seventeen heterofermentative lactobacilli isolated from kefir grains were characterized by molecular methods. Bacterial isolates were identified by amplification of 16S rRNA gene and analysis by Amplified Ribosomal DNA Restriction Analysis (ARDRA), using the restriction enzymes Hae III, Dde I, and Hinf I. ARDRA analysis of lactobacilli isolates showed, for each enzyme used, a same banding pattern between the heterofermentative lactobacilli and the reference strains Lactobacillus kefir JCM 5818 and Lb. kefir ATCC 8007. Other reference lactobacilli and one homofermentative isolate showed differences in at least one of these patterns. The 16S-23S rRNA spacer region was also used to discriminate the bacterial isolates at the species level. The data obtained from the analysis of spacer region confirmed that sequencing of this genome region is a good tool for a reliable identification of members of Lb. kefir species. Genotyping of isolates was performed by Random Amplified Polymorphic DNA (RAPD-PCR) analysis using M13, Coc, ERIC-2 and 1254 primers. Patterns obtained allowed the differentiation of isolates in three groups. The three clusters showed by RAPD-PCR analysis could be correlated with at least three different strains of Lb. kefir species in the group of heterofermentative lactobacilli isolates obtained from Argentinian kefir grains.