Methanotrophs, methanogens and microbial community structure in livestock slurry surface crusts.

AIMS: Crusts forming at the surface of liquid manure (slurry) during storage have been shown to harbour a potential for mitigating CH4 emissions. This study investigated the microbial community in surface crusts, with a focus on micro-organisms related to CH4 metabolism. METHODS AND RESULTS: Microbial communities in four crusts from cattle and swine slurries were investigated using denaturing gradient gel electrophoresis and tag-encoded amplicon pyrosequencing. All crusts had distinct compositions of bacteria and archaea. The genera Methylobacter, Methylomicrobium, Methylomonas, and Methylosarcina of Type I, and Methylocystis of Type II, dominated the methane-oxidizing bacteria (MOB) community, whereas Methanocorpusculum was the predominant methanogen. Higher numbers of operational taxonomic units (OTUs) representing Type I than Type II MOB were found in all crusts. Potential CH4 oxidation rates were determined by incubating crusts with CH4 , and CH4 oxidization was observed in cattle, but not in swine slurry crusts. CONCLUSIONS: Slurry surface crusts harbour a diverse microbial community. Type I MOB are more diverse and abundant than Type II MOB in this environment. The distinct CH4 oxidation rates could be related to microbial compositions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to present the overall microbial community structure in slurry surface crusts. A better understanding of microbial community in surface crusts could support strategies for mitigation of CH4 emissions from livestock manure management.

Specificity and stability of the Acromyrmex-Pseudonocardia symbiosis.

The stability of mutualistic interactions is likely to be affected by the genetic diversity of symbionts that compete for the same functional niche. Fungus-growing (attine) ants have multiple complex symbioses and thus provide ample opportunities to address questions of symbiont specificity and diversity. Among the partners are Actinobacteria of the genus Pseudonocardia that are maintained on the ant cuticle to produce antibiotics, primarily against a fungal parasite of the mutualistic gardens. The symbiosis has been assumed to be a hallmark of evolutionary stability, but this notion has been challenged by culturing and sequencing data indicating an unpredictably high diversity. We used 454 pyrosequencing of 16S rRNA to estimate the diversity of the cuticular bacterial community of the leaf-cutting ant Acromyrmex echinatior and other fungus-growing ants from Gamboa, Panama. Both field and laboratory samples of the same colonies were collected, the latter after colonies had been kept under laboratory conditions for up to 10 years. We show that bacterial communities are highly colony-specific and stable over time. The majority of colonies (25/26) had a single dominant Pseudonocardia strain, and only two strains were found in the Gamboa population across 17 years, confirming an earlier study. The microbial community on newly hatched ants consisted almost exclusively of a single strain of Pseudonocardia while other Actinobacteria were identified on older, foraging ants in varying but usually much lower abundances. These findings are consistent with recent theory predicting that mixtures of antibiotic-producing bacteria can remain mutualistic when dominated by a single vertically transmitted and resource-demanding strain.

Early life treatment with vancomycin propagates Akkermansia muciniphila and reduces diabetes incidence in the NOD mouse.

AIMS/HYPOTHESIS: Increasing evidence suggests that environmental factors changing the normal colonisation pattern in the gut strongly influence the risk of developing autoimmune diabetes. The aim of this study was to investigate, both during infancy and adulthood, whether treatment with vancomycin, a glycopeptide antibiotic specifically directed against Gram-positive bacteria, could influence immune homeostasis and the development of diabetic symptoms in the NOD mouse model for diabetes. METHODS: Accordingly, one group of mice received vancomycin from birth until weaning (day 28), while another group received vancomycin from 8 weeks of age until onset of diabetes. Pyrosequencing of the gut microbiota and flow cytometry of intestinal immune cells was used to investigate the effect of vancomycin treatment. RESULTS: At the end of the study, the cumulative diabetes incidence was found to be significantly lower for the neonatally treated group compared with the untreated group, whereas the insulitis score and blood glucose levels were significantly lower for the mice treated as adults compared with the other groups. Mucosal inflammation was investigated by intracellular cytokine staining of the small intestinal lymphocytes, which displayed an increase in cluster of differentiation (CD)4(+) T cells producing pro-inflammatory cytokines in the neonatally treated mice. Furthermore, bacteriological examination of the gut microbiota composition by pyrosequencing revealed that vancomycin depleted many major genera of Gram-positive and Gram-negative microbes while, interestingly, one single species, Akkermansia muciniphila, became dominant. CONCLUSIONS/INTERPRETATION: The early postnatal period is a critical time for microbial protection from type 1 diabetes and it is suggested that the mucolytic bacterium A. muciniphila plays a protective role in autoimmune diabetes development, particularly during infancy.

Rhizosphere microbial communities associated to rose replant disease: links to plant growth and root metabolites.

Growth depression of Rosa plants at sites previously used to cultivate the same or closely related species is a typical symptom of rose replant disease (RRD). Currently, limited information is available on the causes and the etiology of RRD compared to apple replant disease (ARD). Thus, this study aimed at analyzing growth characteristics, root morphology, and root metabolites, as well as microbial communities in the rhizosphere of the susceptible rootstock Rosacorymbifera 'Laxa' grown in RRD-affected soil from two sites (Heidgraben and Sangerhausen), either untreated or disinfected by γ-irradiation. In a greenhouse bioassay, plants developed significantly more biomass in the γ-irradiated than in the untreated soils of both sites. Several plant metabolites detected in R. corymbifera 'Laxa' roots were site- and treatment-dependent. Although aloesin was recorded in significantly higher concentrations in untreated than in γ-irradiated soils from Heidgraben, the concentrations of phenylalanine were significantly lower in roots from untreated soil of both sites. Rhizosphere microbial communities of 8-week-old plants were studied by sequencing of 16S rRNA, ITS, and cox gene fragments amplified from total community DNA. Supported by microscopic observations, sequences affiliated to the bacterial genus Streptomyces and the fungal genus Nectria were identified as potential causal agents of RRD in the soils investigated. The relative abundance of oomycetes belonging to the genus Pythiogeton showed a negative correlation to the growth of the plants. Overall, the RRD symptoms, the effects of soil treatments on the composition of the rhizosphere microbial community revealed striking similarities to findings related to ARD.

Ecological succession in the vaginal microbiota during pregnancy and birth.

The mother's vaginal microbiota represents the first microbes to which a child is exposed when delivered vaginally. However, little is known about the composition and development of the vaginal microbiota during pregnancy and birth. Here, we analyzed the vaginal microbiota of 57 women in pregnancy week 24, 36 and at birth after rupture of membranes but before delivery, and further compared the composition with that of the gut and airways of the 1-week-old child. The vaginal community structure had dramatic changes in bacterial diversity and taxonomic distribution, yet carried an individual-specific signature. The relative abundance of most bacterial taxa increased stepwise from week 24 of pregnancy until birth, with a gradual decline of Lactobacillus. Mother-to-child vertical transfer, as suggested by sharing, was modest, with the strongest transfer being for Clostridiales followed by Lactobacillales and Enterobacteriales. In conclusion, late gestation is associated with an increase in maternal vaginal microbiota diversity, and vaginal bacteria at birth only modestly predict the composition of the neonatal microbiota.

Cultivation of hard-to-culture subsurface mercury-resistant bacteria and discovery of new merA gene sequences.

Mercury-resistant bacteria may be important players in mercury biogeochemistry. To assess the potential for mercury reduction by two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated natural conditions by using polycarbonate membranes as a growth support and a nonsterile soil slurry as a culture medium. Resistant bacteria were pregrown to microcolony-forming units (mCFU) before being plated on standard medium. Compared to direct plating, culturability was increased up to 2,800 times and numbers of mCFU were similar to the total number of mercury-resistant bacteria in the soils. Denaturing gradient gel electrophoresis analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the preincubation. A total of 25 different 16S rRNA gene sequences were observed, including Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. The diversity of isolates obtained by direct plating included eight different 16S rRNA gene sequences (Alpha- and Betaproteobacteria and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. With phylum-specific merA primers, PCR products were obtained for Alpha- and Betaproteobacteria and Actinobacteria but not for Bacteroidetes and Firmicutes. The similarity to known sequences ranged between 89 and 95%. One of the sequences did not result in a match in the BLAST search. The results illustrate the power of integrating advanced cultivation methodology with molecular techniques for the characterization of the diversity of mercury-resistant populations and assessing the potential for mercury reduction in contaminated environments.

Natural decay process affects the abundance and community structure of Bacteria and Archaea in Picea abies logs.

Prokaryotes colonize decaying wood and contribute to the degradation process, but the dynamics of prokaryotic communities during wood decay is still poorly understood. We studied the abundance and community composition of Bacteria and Archaea inhabiting naturally decaying Picea abies logs and tested the hypothesis that the variations in archaeal and bacterial abundances and community composition are coupled with environmental parameters related to the decay process. The data set comprises >500 logs at different decay stages from five geographical locations in south and central Finland. The results show that Bacteria and Archaea are an integral and dynamic component of decaying wood biota. The abundances of bacterial and archaeal 16S rRNA genes increase as wood decay progresses. Changes in bacterial community composition are clearly linked to the loss of density of wood, while specific fungal-bacterial interactions may also affect the distribution of bacterial taxa in decaying wood. Thaumarchaeota were prominent members of the archaeal populations colonizing decaying wood, providing further evidence of the versatility and cosmopolitan nature of this phylum in the environment. The composition and dynamics of the prokaryotic community suggest that they are an active component of biota that are involved in processing substrates in decaying wood material.

High frequencies of antibiotic resistance genes in infants' meconium and early fecal samples.

The gastrointestinal tract (GIT) microbiota has been identified as an important reservoir of antibiotic resistance genes (ARGs) that can be horizontally transferred to pathogenic species. Maternal GIT microbes can be transmitted to the offspring, and recent work indicates that such transfer starts before birth. We have used culture-independent genetic screenings to explore whether ARGs are already present in the meconium accumulated in the GIT during fetal life and in feces of 1-week-old infants. We have analyzed resistance to ß-lactam antibiotics (BLr) and tetracycline (Tcr), screening for a variety of genes conferring each. To evaluate whether ARGs could have been inherited by maternal transmission, we have screened perinatal fecal samples of the 1-week-old babies' mothers, as well as a mother-infant series including meconium, fecal samples collected through the infant's 1st year, maternal fecal samples and colostrum. Our results reveal a high prevalence of BLr and Tcr in both meconium and early fecal samples, implying that the GIT resistance reservoir starts to accumulate even before birth. We show that ARGs present in the mother may reach the meconium and colostrum and establish in the infant GIT, but also that some ARGs were likely acquired from other sources. Alarmingly, we identified in both meconium and 1-week-olds' samples a particularly elevated prevalence of mecA (>45%), six-fold higher than that detected in the mothers. The mecA gene confers BLr to methicillin-resistant Staphylococcus aureus, and although its detection does not imply the presence of this pathogen, it does implicate the young infant's GIT as a noteworthy reservoir of this gene.

Chromosomal insertion of the entire Escherichia coli lactose operon, into two strains of Pseudomonas, using a modified mini-Tn5 delivery system.

A 12-kb PstI fragment including the entire E. coli lactose operon (lacIPOZYA) was inserted in one copy into the chromosome of Pseudomonas putida, Pseudomonas fluorescens and an E. coli strain with lac- phenotype. This was made possible by improvements of an already existing mini-Tn5 transposon delivery system (de Lorenzo et al., 1990; Herrero et al., 1990), which integrates cloned DNA fragments at random sites on the chromosome of the recipient bacteria in single copies. This has resulted in: (a) the making of two useful low copy-number cloning vectors both with extensive multi-cloning regions flanked by NotI sites needed in the mini-Tn5 delivery system; (b) the generation of E. coli nonlysogenic strains expressing the pi protein thus being capable of maintaining and delivering R6K-based mini-Tn5 vectors to other E. coli strains; (c) the successful insertion of the E. coli lactose operon into the P. fluorescens chromosome giving P. fluorescens the ability to grow on lactose; (d) evidence from Southern blotting that contradicts the assumption that the mini-Tn5 delivery system always creates one-copy inserts. These improvements allow insertion of large DNA fragments encoding highly expressed proteins into the chromosome of a large variety of Gram-negative bacteria including E. coli.

Detection and quantification of tetracyclines by whole cell biosensors.

Three different mini-Tn5 plasmids, containing a tetracycline-inducible promoter, Ptet and a regulatory gene, tetR, in operon fusions with a reporter gene system (lacZYA, luxCDABE or gfp), were constructed. These biosensor constructs responded to low levels of tetracyclines by producing beta-galactosidase, light or green fluorescent protein. They did so in a quantitative manner, thus enabling the quantification of tetracyclines in the immediate surroundings of the biosensor organism. All three constructs were transferred successfully to different gram-negative bacteria by conjugation. An Escherichia coli strain containing the Ptet-lac construct was used to determine oxytetracycline in milk as a demonstration of the application of these biosensors.

Versatile biosensor vectors for detection and quantification of mercury.

Three different whole cell biosensor constructs were made by fusing the mercury inducible promoter, P(mer), and its regulatory gene, merR, from transposon Tn21 with reporter genes luxCDABE, lacZYA, or gfp. In Escherichia coli these biosensor constructs responded to low levels of mercury by producing light, beta-galactosidase or green fluorescent protein, respectively. Since the responses were quantitative, the constructs were used to quantify bioavailable mercury in different environments. The constructs were cloned into mini-Tn5 delivery vectors, thus enabling the transfer of the mer-lux, mer-lac or mer-gfp cassettes to a variety of Gram-negative bacteria. The mer-lux cassette was transferred to a Pseudomonas putida strain, which was used to quantify water-extractable mercury in contaminated soil.

Adaptation of the bacterial community to mercury contamination.

The utilisation of 31 sole carbon sources by bacterial communities of soil in the presence of increasing concentrations of Hg(II) was measured by a colour development assay. The assay was performed on Biolog microtitre plates (Ecoplates) in the presence of Hg(II) and compared to Hg(II)-free Ecoplates. Furthermore, community tolerance to Hg(II) was measured by colour development in microtitre plates supplemented with LB broth and by enumeration of colony-forming units on LB agar plates. Both microtitre plates supplemented with LB and LB agar plates contained increasing concentrations of Hg(II). The difference in substrate utilisation profile, as shown by growth on 31 different carbon substrates in the Ecoplates, suggested an adaptation of the soil community that correlated with the metal exposure level in the soil. Similarly, growth on microtitre plates supplemented with LB and plate-spreading data showed an increased community tolerance with increasing levels of mercury in the soil. Both the multi-function microtitre plate assay (Ecoplate) and the LB broth microtitre plate assay are suitable for evaluating the adaptation of the bacterial community in soil to a heavy metal pollutant.

The Use of Whole-Cell Biosensors to Detect and Quantify Compounds or Conditions Affecting Biological Systems.

A new and promising technique in microbial ecology and environmental biology is the use of whole-cell bacterial biosensors. This minireview describes the use of such biosensors for detection and quantification of various compounds and other conditions affecting bacterial expression of different genes. Three types of biosensors (nonspecific, stress-induced, and specific biosensors) are described including their use in different environments. We present tables of published biosensors, including gene fusions, host organisms, and environments in which they are used. We here describe the use of different reporter genes in the construction of biosensors and discuss their use as tools for monitoring the bioavailability of pollutants and their potential use in studying microbial ecology in general.

Group-Specific PCR Primers to Amplify 24S a-Subunit rRNA Genes from Kinetoplastida (Protozoa) Used in Denaturing Gradient Gel Electrophoresis.

We developed and tested a set of primers for amplification of a region of the 24S a-subunit rRNA genes (24S rDNA) specific to Kinetoplastida (Protozoa). The reverse primer was supplied with a GC rich region in the 5? end in order to make the PCR product suitable for analysis by denaturing gradient gel electrophoresis (DGGE). PCR product was obtained from all the kinetoplastids tested and no PCR product was obtained from any other Eukaryotes or Prokaryotes tested. It was possible to distinguish between all pure cultures of kinetoplastids by denaturing gradient gel electrophoresis in gels ranging from 20% to 60% denaturants. PCR-DGGE analysis of DNA purified from lake sediment revealed approximately 20 bands indicating high kinetoplastid diversity. Direct cloning and sequencing of 24S rDNA sequences retrieved from the lake sediment by PCR also showed high kinetoplastid diversity. Of 43 clones, 27 different sequences were found. Alignments and phylogenetic analysis showed that a majority of the sequences were most closely related to the Bodonidae. Four sequences were closer to the Trypanosomatidae, whereas three sequences fell outside both groups. The PCR-DGGE procedure developed in this study has been shown to be useful for distinguishing between different kinetoplastid species. Thus, it may be a useful tool for evaluating the genetic diversity of this group in environmental samples, e.g., as a result of perturbation. Another possible application of this method is in fast and accurate screening for the presence and identification of pathological parasitic Kinetoplastida from environmental samples and for diagnostics of human and animal infections.

Effects of barley variety and processing methods on feedlot steer performance and carcass characteristics.

An experiment was conducted to evaluate ammoniation and temper processing of two barley varieties of diverse types on feedlot cattle performance and diet digestibility. Steptoe (feed variety) and Klages (malting variety) barleys were processed as dry-rolled (DR); tempered and rolled (TR); tempered, ammoniated, and rolled (AR); and tempered, ammoniated, and fed whole (AW). Crossbred steers (n = 240, initial weight 266 kg) were blocked by weight and randomly assigned to one of eight treatments in a 2 x 4 factorial arrangement. Diets contained 30% barley (DM basis) for the growing phase and 85% (DM basis) for the finishing phase. Growing phase ADG and gain to feed (G/F) were less (P < .05) for AW than for DR, TR, and AR. Average daily gain was less (P < .05) for AW than for TR and AR in the finishing phase. There were no differences (P > .05) in ADG or G/F between DR, TR, and AR during growing or finishing phases. Gain to feed was greater (P < .05) for TR and AR than for AW but not for DR for the total trial. Hot carcass weight, longissimus muscle area, and kidney-pelvic-heart fat were greater (P < .05) for TR and AR than for AW. Total finishing diet ADF digestibility was greater (P < .05) for Steptoe than for Klages (40.5 vs 31.4%, respectively). The DR treatment had the lowest ADF digestibility, whereas AR had the greatest (P < .05). Results suggest that there were no differences in feedlot steer performance due to barley varieties of the same bulk density and that barley grain must be mechanically processed for optimal performance response rather than ammoniated and fed as whole grain.

Genome Sequences of Two Leuconostoc pseudomesenteroides Strains Isolated from Danish Dairy Starter Cultures.

The lactic acid bacterium Leuconostoc pseudomesenteroides can be found in mesophilic cheese starters, where it produces aromatic compounds from, e.g., citrate. Here, we present the draft genome sequences of two L. pseudomesenteroides strains isolated from traditional Danish cheese starters.

Genome Sequence of Leuconostoc mesenteroides subsp. cremoris Strain T26, Isolated from Mesophilic Undefined Cheese Starter.

Leuconostoc is the main group of heterofermentative bacteria found in mesophilic dairy starters. They grow in close symbiosis with the Lactococcus population and are able to degrade citrate. Here we present a draft genome sequence of Leuconostoc mesenteroides subsp. cremoris strain T26.

Characterization of the gut microbiota in leptin deficient obese mice - Correlation to inflammatory and diabetic parameters.

Gut microbiota have been implicated as a relevant factor in the development of type 2 diabetes mellitus (T2DM), and its diversity might be a cause of variation in animal models of T2DM. In this study, we aimed to characterise the gut microbiota of a T2DM mouse model with a long term vision of being able to target the gut microbiota to reduce the number of animals used in experiments. Male B6.V-Lep(ob)/J mice were characterized according to a number of characteristics related to T2DM, inflammation and gut microbiota. All findings were thereafter correlated to one another in a linear regression model. The total gut microbiota profile correlated to glycated haemoglobin, and high proportions of Prevotellaceae and Lachnospiraceae correlated to impaired or improved glucose intolerance, respectively. In addition, Akkermansia muciniphila disappeared with age as glucose intolerance worsened. A high proportion of regulatory T cells correlated to the gut microbiota and improved glucose tolerance. Furthermore, high levels of IL-10, IL-12 and TNF-α correlated to impaired glucose tolerance, blood glucose or glycated haemoglobin. The findings indicate that gut microbiota may contribute to variation in various disease read-outs in the B6.V-Lep(ob)/J model and considering them in both quality assurance and data evaluation for the B6.V-Lep(ob)/J model may have a reducing impact on the inter-individual variation.

Evaluation of two commercial, rapid, ELISA kits testing for scrapie in retro-pharyngeal lymph nodes in sheep.

AIMS: To estimate the number of cases of scrapie that would occur in sheep of different prion protein (PrP) genotypes if scrapie was to become established in New Zealand, and to compare the performance of two commercially available, rapid ELISA kits using ovine retro-pharyngeal lymph nodes (RLN) from non-infected and infected sheep of different PrP genotypes. METHODS: Using published data on the distribution of PrP genotypes within the New Zealand sheep flock and the prevalence of cases of scrapie in these genotypes in the United Kingdom, the annual expected number of cases of scrapie per genotype was estimated, should scrapie become established in New Zealand, assuming a total population of 28 million sheep. A non-infected panel of RLN was collected from 737 sheep from New Zealand that had been culled, found in extremis or died. Brain stem samples were also collected from 131 of these sheep. A second panel of infected samples comprised 218 and 117 RLN from confirmed scrapie cases that had originated in Europe and the United States of America, respectively. All samples were screened using two commercial, rapid, transmissible spongiform encephalopathy ELISA kits: Bio-Rad TeSeE ELISA (ELISA-BR), and IDEXX HerdChek BSE-Scrapie AG Test (ELISA-ID). RESULTS: If scrapie became established in New Zealand, an estimated 596 cases would occur per year; of these 234 (39%) and 271 (46%) would be in sheep carrying ARQ/ARQ and ARQ/VRQ PrP genotypes, respectively. For the non-infected samples from New Zealand the diagnostic specificity of both ELISA kits was 100%. When considering all infected samples, the diagnostic sensitivity was 70.4 (95% CI=65.3-75.3)% for ELISA-BR and 91.6 (95% CI=88.2-94.4)% for ELISA-ID. For the ARQ/ARQ genotype (n=195), sensitivity was 66.2% for ELISA-BR and 90.8% for ELISA-ID, and for the ARQ/VRQ genotype (n=107), sensitivity was 81.3% for ELISA-BR and 98.1% for ELISA-ID. CONCLUSIONS: In this study, the ELISA-ID kit demonstrated a higher diagnostic sensitivity for detecting scrapie in samples of RLN from sheep carrying scrapie-susceptible PrP genotypes than the ELISA-BR kit at comparable diagnostic specificity. CLINICAL RELEVANCE: The diagnostic performance of the ELISA-ID kit using ovine RLN merits the consideration of including this assay in the national scrapie surveillance programme in New Zealand.

Metamobilomics--expanding our knowledge on the pool of plasmid encoded traits in natural environments using high-throughput sequencing.

A metamobilome is defined as a metagenome of circular genetic elements within a certain community. Metagenomic analyses of plasmids provide insights into the composition and structure of environmental plasmid communities. It is a promising method that will provide information about the types of plasmids that are present within environmental samples, and will give overviews about occurrences of plasmids as well as accessory genetic elements carried on these plasmids. A metamobilome library was constructed by combining multiple displacement amplification with pyrosequencing. This method provided a fast, efficient and unbiased strategy to investigate the communal gene pool of circular genetic elements (the metamobilome). We compared our wastewater metamobilome library with a wastewater metagenome library, against chromosomes, plasmids, phages and IS element databases, respectively. This showed that very few strictly chromosomal reads were present in our metamobilome library. Furthermore, data analysis showed that our library was strongly enriched for genes encoding plasmid-selfish traits, such as stability and conjugation, and most strikingly several hundred new putative plasmid replicases have been recovered.