rDNA Profiles Research Articles

Ultra-deep sequencing of 45S rDNA to discern intragenomic diversity in three Chrysodeixis species for molecular identification

Species identification is necessary to prevent introductions of exotic plant pests through global trade. Many of these pests are understudied and lack publicly available DNA sequence data on which rapid molecular identification methods can be based. One such lineage is the genus Chrysodeixis, which includes three species of potential concern for United States trade initiatives: C. includens, C. chalcites, and C. eriosoma. Here we describe a method to generate robust 45S rDNA profiles using long read sequencing in order to clarify evolutionary relationships and develop a real-time PCR identification technique. Such an identification tool will be useful in rapidly differentiating between Chrysodeixis species of quarantine concern where traditional morphological identification methods are insufficient. Molecular methods such as this greatly reduce the time spent identifying each specimen, allow for detection of eDNA, vastly increase throughput, and increase the probability of detection. The methods presented here will be generally adaptable to any understudied lepidopteran taxa that necessitates a molecular diagnostic assay and, with adjustment or testing of the primers, could be applied to any group for which development of rDNA profiles in a benchtop setting would prove useful.

Constitution of mucosa‐associated microbiota in the lower digestive tract does not change in early stage of non‐alcoholic fatty liver disease with fecal dysbiosis

Background and AimRegarding the gut–liver axis, fecal dysbiosis is implicated in the pathogenesis of non‐alcoholic fatty liver disease (NAFLD). The significance of mucosa‐associated microbiota (MAM, which is present in the mucin layer covering the intestinal mucosa) has not been well explored. We aimed to clarify the characteristics of MAM in patients with NAFLD.MethodsMAM were obtained from seven patients with early‐stage NAFLD and seven controls by colonoscopy in five locations (terminal ileum, cecum, ascending and sigmoid colon, and rectum) using mucosal brushes. The microbial 16S rDNA profiles of the MAM and fecal microbiota of patients in the NAFLD and control groups were analyzed.Resultsα‐diversities of fecal microbiota were decreased in patients with NAFLD (observed species, Shannon index, and Chao1: 174.57 vs 134.86, 5.51 vs 4.65, and 206.34 vs 167.91; P = 0.048, 0.067, and 0.087, respectively), and microbial composition analyses by principal coordinate analysis differed between the fecal microbiota of patients with NAFLD and those of controls (permutational analysis of variance [PERMANOVA] of weighted and unweighted: Pseud‐F: 1.4179/P‐value: 0.05 and Pseud‐F: 2.1497/P‐value: 0.049, respectively). However, α‐diversities or microbial composition of MAM in most parts of the intestine did not differ significantly between the NAFLD and control groups. Unclassified Rikenellaceae, Oscillospira, Odoribacter, unclassified clostridiales, and Holdemania were decreased in the feces of patients with NAFLD (determined by linear discriminant analysis effect size), but five (except Holdemania) of the six genera were not decreased in the MAM of these patients.ConclusionIn early‐stage NAFLD, MAM was uniform and relatively stable throughout the intestine, even when fecal dysbiosis appeared.

Abstract 6265: Do epigenetic outliers impact racial disparities in colorectal cancer

Abstract African Americans (AA) have a higher incidence and mortality rate for colorectal cancer (CRC) compared to Caucasians. DNA methylation profiling on normal colon mucosa from 77 CRC patients and 68 patients without cancer (controls) of AA and Caucasian descent identified a distinct subgroup of CRC patients (11AA and 4 Caucasians) who are statistical “outliers” at a large number of CpGs. Such highly epigenetically disrupted CRC patients are termed “Outlier Methylation Phenotype” (OMP). Comparison of methylome profiles of CRC patients and controls revealed that AA CRC patients have greater disruption of the normal colon methylome than do Caucasian patients. A large fraction of the overall methylation differences found between CRC patients and controls is attributable to OMPs. Estimation of epigenetic ages shows that OMPs have lower epigenetic ages compared to their chronological ages. In other words, OMPs undergo epigenetic age deceleration. Comparison of normal colon mucosa transcription profiles of OMP cancer patients with those of non-OMP cancer patients indicates genes whose promoters are hypermethylated in the OMP patients are also transcriptionally down-regulated, and that many of the genes most affected are involved in interactions between epithelial cells, the mucus layer, and the microbiome. Analysis of 16S RDNA profiles suggests that normal colon mucosa of OMPs are enriched in bacterial genera associated with CRC risk, advanced tumor stage, chronic intestinal inflammation, malignant transformation, nosocomial infections and KRAS mutations. In conclusion, our study suggests a distinct epigenetically disrupted OMP group in CRC that are responsible for disrupted methylome, altered gene expression and microbial dysbiosis. Furthermore, a higher frequency of OMPs among African Americans (11/42) than among Caucasians (5/35) may contribute to observed racial disparities in CRC incidence and outcome. Citation Format: Jayashri Ghosh, Bryant M. Schultz, Joe Chan, Claudia Wultsch, Imad Shureiqi, Stephanie Chow, Ahmet Doymaz, Sophia Varriano, Melissa Driscoll, Jen Muse, Frida E. Kleiman, Konstantinos Krampis, Jean-Pierre J. Issa, Carmen Sapienza. Do epigenetic outliers impact racial disparities in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6265.

Diversity and Potential Function of Prokaryotic and Eukaryotic Communities from Different Mangrove Sediments

Mangrove trees generally play important roles in protecting intertidal ecosystems. The mangrove root-associated sediments provide a repertoire of microbial communities that contribute to pivotal ecological functions in the system. In the present study, we used the high-throughput sequencing and PICRUSt-predicted functional information (based on 16S/18S rDNA profiles) to investigate the bacterial, archaeal, and fungal communities in two mangrove systems, located in the estuary of the Jiulong River (China), with different contaminated conditions and frequencies of human activity. Diverse distribution patterns for microbial communities were observed in six sediment samples collected from the two survey areas, which were found to be related mainly to the substrates in mangrove sediments. The sediments were predominated by relatively higher ratios of heterotrophic bacteria that participated in the degradation of organic matters, including phylum of Chloroflexi, Acidobacteriota, Desulfobacterota, and Proteobacteria. In addition, Crenarchaeota and Ascomycota presented the highest abundances of archaea and fungi, respectively. The relatively high concentrations of calcium, nitrogen, magnesium, and phosphorus in mangrove sediments correlated significantly with the microbial communities. In addition, although the potential functions were similar in the two sites based on COG and KEGG pathways, the abundances of enzymes involved in the degradation processes of cellulose and hemicellulose and the metabolism of nitrogen and sulfur presented distinctions. These results provide insights into the environmental conditions shaping microbial assemblies of the mangrove sediments under the impacts of human activities; for instance, a more abundant amount of calcium was found in urban areas in this study.

Performance of distinct microbial based solutions in a Campylobacter infection challenge model in poultry

BackgroundAntibiotic growth promoters (AGPs) are commonly used within poultry production to improve feed conversion, bird growth, and reduce morbidity and mortality from clinical and subclinical diseases. Due to the association between AGP usage and rising antimicrobial resistance, the industry has explored new strategies including the use of probiotics and other microbial-based interventions to promote the development of a healthy microbiome in birds and mitigate against infections associated with food safety and food security. While previous studies have largely focused on the ability of probiotics to protect against Clostridium perfringens and Salmonella enterica, much less is known concerning their impact on Campylobacter jejuni, a near commensal of the chicken gut microbiome that nevertheless is a major cause of food poisoning in humans.ResultsHere we compare the efficacy of four microbial interventions (two single strain probiotics, the bacterium—Pediococcus acidilactici, and the yeast—Saccharomyces cerevisiae boulardii; and two complex, competitive exclusion, consortia—Aviguard and CEL) to bacitracin, a commonly used AGP, to modulate chicken gut microbiota and subsequently impact C. jejuni infection in poultry. Cecal samples were harvested at 30- and 39-days post hatch to assess Campylobacter burden and examine their impact on the gut microbiota. While the different treatments did not significantly decrease C. jejuni burden relative to the untreated controls, both complex consortia resulted in significant decreases relative to treatment with bacitracin. Analysis of 16S rDNA profiles revealed a distinct microbial signature associated with each microbial intervention. For example, treatment with Aviguard and CEL increased the relative abundance of Bacteroidaceae and Rikenellaceae respectively. Furthermore, Aviguard promoted a less complex microbial community compared to other treatments.ConclusionsDepending upon the individual needs of the producer, our results illustrate the potential of each microbial interventions to serve flock-specific requirements.

Determination of bacterial population and the presence of pesticide residues from some Cameroonian smoked and dried fish

The objective of this study was to identify the dominant bacterial flora linked to the hygiene of dried and smoked fish in different geographical locations and to determine the presence of pesticide residues on the processed fish. Fish species were listed and collected. The bacterial ecology of dried and smoked fish from the northern zone of Cameroon was probed using a molecular technique employing variable regions of 16S rDNA profiles generated by PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis). The bacterial species were obtained by sequencing the PCR-DGGE bands and the pesticide residues by Chromatography coupled with mass spectrometry (GC-MS / MS), tested on 11 fish samples. Fifty three percent of the fish were smoked. Three types of pesticides have been identified on the dried fish samples. Among them, there were Cypermethrin (15 to 3600 µg/kg), Chlorpyrifos (19 to 8800 µg/kg) and Profenophos (62 to 92 µg/kg). The analysis of 16S rDNA profile and DNA sequencing revealed the presence of 32 bacterial species grouped into 22 genera. Dried fish were the most contaminated. Species such as Vagococcus carniphilus, Kurthia gibsonii, Lysinibacillus endophyticus, Macrococcus caseolyticus and Bacillus atrophaeus were present in the samples from all the locations. On a general note, the fish samples are of poor microbiological and sanitary quality.

Mastomys natalensis (Smith, 1834) as a natural host for Schistosoma haematobium (Bilharz, 1852) Weinland, 1858 x Schistosoma bovis Sonsino, 1876 introgressive hybrids

Cercarial emission of schistosomes is a determinant in the transmission to the definitive host and constitutes a good marker to identify which definitive host is responsible for transmission, mainly in introgressive hybridization situations. Our goal was to test the hypothesis that micro-mammals play a role in Schistosoma haematobium, S. bovis, and/or S. haematobium x S. bovis transmission. Small mammal sampling was conducted in seven semi-lacustrine villages of southern Benin. Among the 62 animals trapped, 50 individuals were investigated for Schistosoma adults and eggs: 37 Rattus rattus, 3 Rattus norvegicus, 9 Mastomys natalensis, and 1 Crocidura olivieri. Schistosoma adults were found in four R. rattus and two M. natalensis, with a local prevalence reaching 80% and 50%, respectively. Two cercarial chronotypes were found from Bulinus globosus experimentally infected with miracidia extracted from naturally infected M. natalensis: a late diurnal and nocturnal chronotype, and an early diurnal, late diurnal, and nocturnal chronotype. The cytochrome C oxidase subunit I mtDNA gene of the collected schistosomes (adults, miracidia, and cercariae) belonged to the S. bovis clade. Eleven internal transcribed spacer rDNA profiles were found; four belonged to S. bovis and seven to S. haematobium x S. bovis. These molecular results together with the observed multi-peak chronotypes add M. natalensis as a new host implicated in S. haematobium x S. bovis transmission. We discuss the origin of the new chronotypes which have become more complex with the appearance of several peaks in a 24-h day. We also discuss how the new populations of offspring may optimize intra-host ecological niche, host spectrum, and transmission time period.

Identification of yeasts present in artisanal yoghurt and traditionally fermented milks consumed in the northern part of Cameroon

Abstract Bacteria generally ferment milk but, sometimes, yeasts are found in fermented milks. The presence of these yeasts in the microbial community of some fermented milks could be intentional or accidental. The Diversity of yeasts in the products was investigated using a molecular technique employing variable regions of 26S rDNA profiles generated by PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis). Two types of samples: artisanal or handcraft yoghurt and traditionally fermented milks were collected in three towns of the three regions of the northern part of Cameroon. Firstly, a comparison was made between the 16 traditionally fermented milks collected in Maroua, Garoua and Ngaoundre each of the 3 regions. Secondly, it was between 26 artisanal fermented milks of each region and finally, between the two types of products. The different PCR-DGGE 26S rDNA profiles obtained were analyzed and DNA sequencing was used to compare yeasts from each method of production. Twelve (12) species of Yeasts were identified as: Malassezia globosa, Hanseniaspora uvarum, Galactomyces candidum, Candida tropicalis, Aureobasidium pullulans, Torulaspora globosa, Saccharomyces cerevisiae/paradoxus, Pichia kluyveri, Candida parapsilosis, Torulaspora delbrueckii, Kluyveromyces marxianus, Candida orthopsilosis and Pseudozyma sp. Yeast diversity was higher for artisanal fermented milks (yoghurt) with at least 10 species, while for traditionally non packed fermented milks only 5 species were identified with a predominance strain of Galactomyces candidum, Candida parapsilosis, Torulaspora delbrueckii, Saccharomyces cerevisiae/paradoxus and Kluyveromyces marxianus. The different species of yeast might be introduced accidentally in artisanal yoghurt; however, for traditionally fermented milks their presence might be associated to the starter.

CowPI: A Rumen Microbiome Focussed Version of the PICRUSt Functional Inference Software.

Metataxonomic 16S rDNA based studies are a commonplace and useful tool in the research of the microbiome, but they do not provide the full investigative power of metagenomics and metatranscriptomics for revealing the functional potential of microbial communities. However, the use of metagenomic and metatranscriptomic technologies is hindered by high costs and skills barrier necessary to generate and interpret the data. To address this, a tool for Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was developed for inferring the functional potential of an observed microbiome profile, based on 16S data. This allows functional inferences to be made from metataxonomic 16S rDNA studies with little extra work or cost, but its accuracy relies on the availability of completely sequenced genomes of representative organisms from the community being investigated. The rumen microbiome is an example of a community traditionally underrepresented in genome and sequence databases, but recent efforts by projects such as the Global Rumen Census and Hungate 1000 have resulted in a wide sampling of 16S rDNA profiles and almost 500 fully sequenced microbial genomes from this environment. Using this information, we have developed “CowPI,” a focused version of the PICRUSt tool provided for use by the wider scientific community in the study of the rumen microbiome. We evaluated the accuracy of CowPI and PICRUSt using two 16S datasets from the rumen microbiome: one generated from rDNA and the other from rRNA where corresponding metagenomic and metatranscriptomic data was also available. We show that the functional profiles predicted by CowPI better match estimates for both the meta-genomic and transcriptomic datasets than PICRUSt, and capture the higher degree of genetic variation and larger pangenomes of rumen organisms. Nonetheless, whilst being closer in terms of predictive power for the rumen microbiome, there were differences when compared to both the metagenomic and metatranscriptome data and so we recommend, where possible, functional inferences from 16S data should not replace metagenomic and metatranscriptomic approaches. The tool can be accessed at http://www.cowpi.org and is provided to the wider scientific community for use in the study of the rumen microbiome.

Sediment bacteria in an urban stream: Spatiotemporal patterns in community composition

Sediment bacterial communities play a critical role in biogeochemical cycling in lotic ecosystems. Despite their ecological significance, the effects of urban discharge on spatiotemporal distribution of bacterial communities are understudied. In this study, we examined the effect of urban discharge on the spatiotemporal distribution of stream sediment bacteria in a northeast Ohio stream. Water and sediment samples were collected after large storm events (discharge > 100 m) from sites along a highly impacted stream (Tinkers Creek, Cuyahoga River watershed, Ohio, USA) and two reference streams. Although alpha (α) diversity was relatively constant spatially, multivariate analysis of bacterial community 16S rDNA profiles revealed significant spatial and temporal effects on beta (β) diversity and community composition and identified a number of significant correlative abiotic parameters. Clustering of upstream and reference sites from downstream sites of Tinkers Creek combined with the dominant families observed in specific locales suggests that environmentally-induced species sorting had a strong impact on the composition of sediment bacterial communities. Distinct groupings of bacterial families that are often associated with nutrient pollution (i.e., Comamonadaceae, Rhodobacteraceae, and Pirellulaceae) and other contaminants (i.e., Sphingomonadaceae and Phyllobacteriaceae) were more prominent at sites experiencing higher degrees of discharge associated with urbanization. Additionally, there were marked seasonal changes in community composition, with individual taxa exhibiting different seasonal abundance patterns. However, spatiotemporal variation in stream conditions did not affect bacterial community functional profiles. Together, these results suggest that local environmental drivers and niche filtering from discharge events associated with urbanization shape the bacterial community structure. However, dispersal limitations and interactions among other species likely play a role as well.

Cytogenetics of Two Onychostoma Species in Taiwan by Ag-NOR and 18S rDNA Profiles.

Chiao-Chuan Han, Tsair-Bor Yen, Nian-Cih Chen, and Mei-Chen Tseng (2017) Both Onychostoma barbatulum and O. alticorpus are primary freshwater fish in Taiwan. The former has been developed as an aquaculture species with high economic value, while the latter is a native endemic species in Taiwan. Understanding the cytogenetic information of these two species is necessary for their selected breeding, recovery, and management. In this study, Giemsa staining, silver-binding nucleolar organizer region (Ag-NOR), C-banding, and fluorescence in situ hybridization (FISH) with 18S ribosomal (r)DNA probes were used to analyze the cytogenetic characteristics. Results of Giemsa staining showed that the two Onychostoma species shared the same number of chromosomes, 2n = 50. Respective karyotype formulas of the female and male were 10 m + 22 sm + 10 st + 8 t and 11 m + 22 sm + 10 st + 7 t in O. barbatulum, and 14 m + 18 sm + 8 st + 10 t and 15 m + 18 sm + 8 st + 9 t in O. alticorpus. Karyotypes of both species showed a pair of heteromorphic chromosomes in male fish. Their sex determination should be the XX/XY system. Two pairs of Ag-NORs were found in O. barbatulum, but only one pair occurred in O. alticorpus. C-banding areas were observed on centromeres or telomeres of some chromosomes. FISH revealed different cytogenetic characters between these two species. The above cytogenetic information will contribute to species identification, population recovery, and advantages for breeding and management in the future.

The functional potential and active populations of the pit mud microbiome for the production of Chinese strong-flavour liquor.

SummaryThe popular distilled Chinese strong‐flavour liquor (CSFL) is produced by solid fermentation in the ground pit. Microbes inhabiting in the pit mud (PM) on the walls of the fermentation pit are responsible for the production of caproic acid (CA) that determines the quality of CSFL to a large degree. However, little is known about the active microbial populations and metabolic potential of the PM microbiome. Here, we investigated the overall metabolic features of the PM microbiome and its active microbial components by combining metagenomics and MiSeq‐sequencing analyses of the 16S rRNA genes from DNA and RNA (cDNA). Results showed that prokaryotes were predominant populations in the PM microbiome, accounting for 95.3% of total metagenomic reads, while eukaryotic abundance was only 1.8%. The dominant prokaryotic phyla were Firmicutes, Euryarchaeota, Bacteroidetes, Actinobacteria and Proteobacteria, accounting for 48.0%, 19.0%, 13.5%, 2.5% and 2.1% of total metagenomic reads respectively. Most genes encoding putative metabolic pathways responsible for the putative CA production via chain elongation pathway were detected. This indicated that the PM microbiome owned functional potential for synthesizing CA from ethanol or lactate. Some key genes encoding enzymes involved in hydrogenotrophic and acetoclastic methanogenesis pathways were detected in the PM metagenome, suggesting the possible occurrence of interspecies hydrogen transfer between CA‐producing bacteria and methanogens. The 16S rDNA and 16S rRNA profiles showed that the Clostridial cluster IV, Lactobacillus, Caloramator, Clostridium, Sedimentibacter, Bacteroides and Porphyromonas were active populations in situ, in which Clostridial cluster IV and Clostridium were likely involved in the CA production. This study improved our understandings on the active populations and metabolic pathways of the PM microbiome involved in the CA synthesis in the CSFL fermentation.

Metagenomic rDNA Profiles of the Oral Microbiome in Healthy Subjects Are Distinctive Compared to That in Smokers and Disease Subjects Despite High Variations between Subjects

The vital role of oral microbiome in the well-being of humans is only beginning to be unraveled. Employing a rigorous analysis of PCR-restriction fragment length polymorphisms (PCR-RFLP) and DNA fingerprints from denatured gradient gel electrophoresis (PCR-DGGE) of the 16S rDNA gene in metagenomic samples, this study evaluated the stability of the oral microbiome and contrasted the PCR-DGGE profiles of subjects belonging to three groups—healthy, smokers and oral diseases; in search of distinctive patterns predictive of each group. The DNA band size, intensity and profile generated by three restriction enzymes from a 1500 bp amplicon showed a fairly stable microbial community structure (P H' = 0.99 ± 0.12; S = 2.87 ± 0.75) and oral disease mouths (H' = 1.06 ± 0.085; S = 2.86 ± 0.31), than in healthy subjects (H' = 0.926 ± 0.07; S = 1.79 ± 0.56) suggesting that smoking is associated with a microbial community shift towards the structure found in poor oral health. It is clear that the human oral bacteria symbionts are not all random colonizers. Rather some of them constitute a fragile stable dynamic community whose disturbance could lead to disease or be indicative of disease. Understanding the dynamics of the bacterial community structure in health and disease states is a prerequisite to developing effective preventive healthcare and rapid diagnosis of diseases.

Imbalance of bacteriome profiles within the Finnish Diabetes Prediction and Prevention study: Parallel use of 16S profiling and virome sequencing in stool samples from children with islet autoimmunity and matched controls.

We set out to explore associations between the stool bacteriome profiles and early-onset islet autoimmunity, taking into account the interactions with the virus component of the microbiome. Serial stool samples were longitudinally collected from 18 infants and toddlers with early-onset islet autoimmunity (median age 17.4 months) followed by type 1 diabetes, and 18 tightly matched controls from the Finnish Diabetes Prediction and Prevention (DIPP) cohort. Three stool samples were analyzed, taken 3, 6, and 9 months before the first detection of serum autoantibodies in the case child. The risk of islet autoimmunity was evaluated in relation to the composition of the bacteriome 16S rDNA profiles assessed by mass sequencing, and to the composition of DNA and RNA viromes. Four operational taxonomic units were significantly less abundant in children who later on developed islet autoimmunity as compared to controls-most markedly the species of Bacteroides vulgatus and Bifidobacterium bifidum. The alpha or beta diversity, or the taxonomic levels of bacterial phyla, classes or genera, showed no differences between cases and controls. A correlation analysis suggested a possible relation between CrAssphage signals and quantities of Bacteroides dorei. No apparent associations were seen between development of islet autoimmunity and sequences of yet unknown origin. The results confirm previous findings that an imbalance within the prevalent Bacteroides genus is associated with islet autoimmunity. The detected quantitative relation of the novel "orphan" bacteriophage CrAssphage with a prevalent species of the Bacteroides genus may exemplify possible modifiers of the bacteriome.

Nuclear rDNA instability inin vitro-generated plants is amplified after sexual reproduction with conspecific wild individuals

Using micropropagation through tissue culture has become the most used approach worldwide for mass production for the conservation of endangered species. However, the screening of somaclonal variations generated using in vitro culture is usually restricted to the first generation of micropropagated plants, when they have not yet been released in the field. Accordingly, the fate of genetically modified regenerants after sexual reproduction is usually not assessed and changes in the genetic structures of species are unknown. In this work, we assess the cytogenetic stability of two rDNA gene families in the offspring of experimental crosses between accessions generated after in vitro culture and wild individuals of Cistus heterophyllus (Cistaceae). The cytogenetic rDNA profiles (45S rDNA, 5S rDNA) of 118 accessions including wild and in vitro micropropagated individuals and bi-directional artificial crosses between wild and in vitro-generated plants were assessed by fluorescence in situ hybridization (FISH) and Ag-NOR staining. Plants regenerated by micropropagation showed a lower size of the FISH signals in a 45S rDNA site, but this condition was not present in the wild accessions. Three new cytogenetic and cytological variants were present in 36% of the experimental progeny, involving the amplification of one additional 45S rDNA site and the presence of heteromorphic nucleoli. rDNA-based genomic instability was present after sexual reproduction between wild and in vitro-generated plants. The results of this study discourage the use of micropropagated materials for plant conservation unless comprehensive surveys of the genetic integrity and stability of the regenerants are performed after crossing between wild and micropropagated plants.

Discrimination of post-harvest coffee processing methods by microbial ecology analyses

Two main types of processing are applied to coffee: the dry and wet processes. The aim of this study was to measure the impact of post-harvest processing on coffee microbial ecology and search for microbial markers of process discrimination. Coffee samples were collected in three farms in two locations of West Cameroon (Bafoussam and Dschang). Samples of Coffea arabica were collected along wet and dry methods and Coffea canephora samples were collected only during treatment using the dry method. Bacterial and fungal communities present on coffee beans were determined by using a molecular method PCR-DGGE (Denaturing Gradient Gel Electrophoresis) generating 16S rDNA and 26S rDNA profiles for bacteria and fungi. Results showed that microbial ecology structures were different depending on three main parameters: i) the method applied, ii) the coffee species and iii) their geographical origin. Statistical analysis applied allowed to measure the impact of all parameters and showed that the method was the major parameter influencing microbial communities' structure. Microbial DNA markers that appear specific of coffee origin, species or treatment were identified, such as lactic acid bacteria (LAB) which were detected only during wet process. They are specific of the fermentation step. DNA from Wallemia sp. was only detected in Robusta coffee beans (Coffea canephora). The study of microbial communities at the molecular level appears as a promising approach to discriminate between coffee processing types.

Molecular diversity of berseem (<italic>Trifolium alexandrinum</italic> L.) rhizobia isolated from Haryana soil

Total of forty rhizobia were isolated from nodule samples of berseem crop from farmers’ fields representing 17 villages of Haryana state, India. All the isolates were Gram-negative small rods and authenticated as rhizobia by plant infectivity test. Twenty nine rhizobial isolates on the basis of good nodulation characteristics were further selected for molecular diversity study. The amplified PCR product of genomic DNA of all the 29 rhizobia was digested with HaeIII restriction enzyme which resulted in polymorphic bands. Dendrogram based on RFLP of 16S rDNA profiles showed that all the 29 isolates were distributed in two major groups with different subgroups. A total of 7 biotypes were formed at 80% level of similarity by considering each cluster as rhizobial biotype, and out of these, two biotypes (1<sup>st</sup> and 5<sup>th</sup>) were found to be most prevalent in all the three districts studied. So, the rhizobia belonging to these two biotypes may be used as biofertilizer in these three districts. Moreover, isolates from same nodule were not 100% similar. It indicated that considerable diversity was present among berseem rhizobial isolates

Soil fungal community shift evaluation as a potential cadaver decomposition indicator

Fungi metabolise organic matter in situ and so alter both the bio-/physico-chemical properties and microbial community structure of the ecosystem. In particular, they are responsible reportedly for specific stages of decomposition. Therefore, this study aimed to extend previous bacteria-based forensic ecogenomics research by investigating soil fungal community and cadaver decomposition interactions in microcosms with garden soil (20kg, fresh weight) and domestic pig (Sus scrofa domesticus) carcass (5kg, leg). Soil samples were collected at depths of 0–10cm, 10–20cm and 20–30cm on days 3, 28 and 77 in the absence (control −Pg) and presence (experimental +Pg) of Sus scrofa domesticus and used for total DNA extraction and nested polymerase chain reaction and denaturing gradient gel electrophoresis (PCR–DGGE) profiling of the 18S rRNA gene. The Shannon–Wiener (H′) community diversity indices were 1.25±0.21 and 1.49±0.30 for the control and experimental microcosms, respectively, while comparable Simpson species dominance (S) values were 0.65±0.109 and 0.75±0.015. Generally, and in contrast to parallel studies of the bacterial 16S rRNA and 16S rDNA profiles, statistical analysis (t-test) of the 18S dynamics showed no mathematically significant shifts in fungal community diversity (H′; p=0.142) and dominance (S; p=0.392) during carcass decomposition, necessitating further investigations.

New traceability strategies based on a biological Bar Code by PCR-DGGE using bacterial and yeast communities for determining farming type of peach

IntroductionEU Regulations continue to be strict in order to allow the highest degree of insurance in foodstuffs' safety and quality. The traceability and labeling of imported products in EU still remain a valuable issue (EU-Regulation 178/2002). One of the most important concerns of the customers is the product traceability, which is defined as the ability to screen the whole history or geographical origin or farming type of any food by certified methodologies. In this regard, we proposed to study the link between microbial ecology and farming type of foodstuffs via a unique molecular analytical technique which is followed by an image analysis. PurposeA unique molecular analytical tool using 16S rDNA and 26S rDNA profiles generated by “PCR-DGGE” was used to detect the variation in bacterial and yeast communities respectively of peaches from Jordan (control fruit, organic farming and conventional farming). ResultsWhen the 16S and 26S rDNA profiles were analyzed using image and multivariate analysis, certain microbiota were detected on peach fruits originating from Jordan. The resulted band profiles of bacteria and yeasts from different farming types were specific and could be used as a bar-code to discriminate the fruits' farming type. Significance of the paperThis method could be considered as a new traceability technique which provides fruits with a unique biological bar-code and permits the possibility to control the farming type of foodstuffs.

Microbial dynamics during azo dye degradation in a UASB reactor supplied with yeast extract.

The present work aimed to investigate the microbial dynamics during the anaerobic treatment of the azo dye blue HRFL in bench scale upflow anaerobic sludge bed (UASB) reactor operated at ambient temperature. Sludge samples were collected under distinct operational phases, when the reactor were stable (low variation of color removal), to assess the effect of glucose and yeast extract as source of carbon and redox mediators, respectively. Reactors performance was evaluated based on COD (chemical oxygen demand) and color removal. The microbial dynamics were investigated by PCR-DGGE (Polimerase Chain Reaction - Denaturing Gradient of Gel Electrophoresis) technique by comparing the 16S rDNA profiles among samples. The results suggest that the composition of microorganisms changed from the beginning to the end of the reactor operation, probably in response to the presence of azo dye and/or its degradation byproducts. Despite the highest efficiency of color removal was observed in the presence of 500 mg/L of yeast extract (up to 93%), there were no differences regarding the microbial profiles that could indicate a microbial selection by the yeast extract addition. On the other hand Methosarcina barkeri was detected only in the end of operation when the best efficiencies on color removal occurred. Nevertheless the biomass selection observed in the last stages of UASB operation is probably a result of the washout of the sludge in response of accumulation of aromatic amines which led to tolerant and very active biomass that contributed to high efficiencies on color removal.