Ramiro Vilchez-Vargas

Analysis of the microbial gene landscape and transcriptome for aromatic pollutants and alkane degradation using a novel internally calibrated microarray system

Despite various efforts to develop tools to detect and compare the catabolic potential and activity for pollutant degradation in environmental samples, there is still a need for an open-source, curated and reliable array method. We developed a custom array system including a novel normalization strategy that can be applied to any microarray design, allowing the calculation of the reliability of signals and make cross-experimental comparisons. Array probes, which are fully available to the scientific community, were designed from knowledge-based curated databases for key aromatic catabolic gene families and key alkane degradation genes. This design assigns signals to the respective protein subfamilies, thus directly inferring function and substrate specificity. Experimental procedures were optimized using DNA of four genome sequenced biodegradation strains and reliability of signals assessed through a novel normalization procedure, where a plasmid containing four artificial targets in increased copy numbers and co-amplified with the environmental DNA served as an internal calibration curve. The array system was applied to assess the catabolic gene landscape and transcriptome of aromatic contaminated environmental samples, confirming the abundance of catabolic gene subfamilies previously detected by functional metagenomics but also revealing the presence of previously undetected catabolic groups and specifically their expression under pollutant stress.

A poke into the diversity and associations within human anterior nare microbial communities

The anterior nares are the major reservoir for Staphylococcus aureus in humans, where nasal carriage has a crucial function as a source for invasive infections. Despite various investigations on aerobic community members based on traditional cultivation methods, little is known on the overall microbial composition and complex in situ interactions, but such knowledge is highly warranted for effective S. aureus control strategies. As assessed using advanced culture-independent approaches, this study provides a comprehensive survey of the anterior nare bacterial community of 40 individuals. Previously undiscovered co-colonization patterns and natural variations in species composition were revealed and provide insights for future intervention strategies for the control of health-care- and community-associated S. aureus infections.

Characterisation of the human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 region of the 16S rRNA gene.

Background. It is widely assumed that the uterine cavity in non-pregnant women is physiologically sterile, also as a premise to the long-held view that human infants develop in a sterile uterine environment, though likely reflecting under-appraisal of the extent of the human bacterial metacommunity. In an exploratory study, we aimed to investigate the putative presence of a uterine microbiome in a selected series of non-pregnant women through deep sequencing of the V1-2 hypervariable region of the 16S ribosomal RNA (rRNA) gene. Methods. Nineteen women with various reproductive conditions, including subfertility, scheduled for hysteroscopy and not showing uterine anomalies were recruited. Subjects were highly diverse with regard to demographic and medical history and included nulliparous and parous women. Endometrial tissue and mucus harvesting was performed by use of a transcervical device designed to obtain endometrial biopsy, while avoiding cervicovaginal contamination. Bacteria were targeted by use of a barcoded Illumina MiSeq paired-end sequencing method targeting the 16S rRNA gene V1-2 region, yielding an average of 41,194 reads per sample after quality filtering. Taxonomic annotation was pursued by comparison with sequences available through the Ribosomal Database Project and the NCBI database. Results. Out of 183 unique 16S rRNA gene amplicon sequences, 15 phylotypes were present in all samples. In some 90% of the women included, community architecture was fairly similar inasmuch B. xylanisolvens, B. thetaiotaomicron, B. fragilis and an undetermined Pelomonas taxon constituted over one third of the endometrial bacterial community. On the singular phylotype level, six women showed predominance of L. crispatus or L. iners in the presence of the Bacteroides core. Two endometrial communities were highly dissimilar, largely lacking the Bacteroides core, one dominated by L. crispatus and another consisting of a highly diverse community, including Prevotella spp., Atopobium vaginae, and Mobiluncus curtisii. Discussion. Our findings are, albeit not necessarily generalizable, consistent with the presence of a unique microbiota dominated by Bacteroides residing on the endometrium of the human non-pregnant uterus. The transcervical sampling approach may be influenced to an unknown extent by endocervical microbiota, which remain uncharacterised, and therefore warrants further validation. Nonetheless, consistent with our understanding of the human microbiome, the uterine microbiota are likely to have a previously unrecognized role in uterine physiology and human reproduction. Further study is therefore warranted to document community ecology and dynamics of the uterine microbiota, as well as the role of the uterine microbiome in health and disease.

Inoculum selection is crucial to ensure operational stability in anaerobic digestion

Anaerobic digestion is considered a key technology for the future bio-based economy. The microbial consortium carrying out the anaerobic digestion process is quite complex, and its exact role in terms of “elasticity”, i.e., the ability to rapidly adapt to changing conditions, is still unknown. In this study, the role of the initial microbial community in terms of operational stability and stress tolerance was evaluated during a 175-day experiment. Five different inocula from stable industrial anaerobic digesters were fed a mixture of waste activated sludge and glycerol. Increasing ammonium pulses were applied to evaluate stability and stress tolerance. A different response in terms of start-up and ammonium tolerance was observed among the different inocula. Methanosaetaceae were the dominant acetoclastic methanogens, yet, Methanosarcinaceae increased in abundance at elevated ammonium concentrations. A shift from a Firmicutes to a Proteobacteria dominated bacterial community was observed in failing digesters. Methane production was strongly positively correlated with Methanosaetaceae, but also with Bacteria related to Anaerolinaceae, Clostridiales, and Alphaproteobacteria. Volatile fatty acids were strongly positively correlated with Betaproteobacteria and Bacteroidetes, yet ammonium concentration only with Bacteroidetes. Overall, these results indicate the importance of inoculum selection to ensure stable operation and stress tolerance in anaerobic digestion.

Optimized Cryopreservation of Mixed Microbial Communities for Conserved Functionality and Diversity

The use of mixed microbial communities (microbiomes) for biotechnological applications has steadily increased over the past decades. However, these microbiomes are not readily available from public culture collections, hampering their potential for widespread use. The main reason for this lack of availability is the lack of an effective cryopreservation protocol. Due to this critical need, we evaluated the functionality as well as the community structure of three different types of microbiomes before and after cryopreservation with two cryoprotective agents (CPA). Microbiomes were selected based upon relevance towards applications: (1) a methanotrophic co-culture (MOB), with potential for mitigation of greenhouse gas emissions, environmental pollutants removal and bioplastics production; (2) an oxygen limited autotrophic nitrification/denitrification (OLAND) biofilm, with enhanced economic and ecological benefits for wastewater treatment, and (3) fecal material from a human donor, with potential applications for fecal transplants and pre/probiotics research. After three months of cryopreservation at −80°C, we found that metabolic activity, in terms of the specific activity recovery of MOB, aerobic ammonium oxidizing bacteria (AerAOB) and anaerobic AOB (AnAOB, anammox) in the OLAND mixed culture, resumes sooner when one of our selected CPA [dimethyl sulfoxide (DMSO) and DMSO plus trehalose and tryptic soy broth (DMSO+TT)] was added. However, the activity of the fecal community was not influenced by the CPA addition, although the preservation of the community structure (as determined by 16S rRNA gene sequencing) was enhanced by addition of CPA. In summary, we have evaluated a cryopreservation protocol that succeeded in preserving both community structure and functionality of value-added microbiomes. This will allow individual laboratories and culture collections to boost the use of microbiomes in biotechnological applications.

High-Resolution Taxonomic Profiling of the Subgingival Microbiome for Biomarker Discovery and Periodontitis Diagnosis

ABSTRACT The oral microbiome plays a key role for caries, periodontitis, and systemic diseases. A method for rapid, high-resolution, robust taxonomic profiling of subgingival bacterial communities for early detection of periodontitis biomarkers would therefore be a useful tool for individualized medicine. Here, we used Illumina sequencing of the V1-V2 and V5-V6 hypervariable regions of the 16S rRNA gene. A sample stratification pipeline was developed in a pilot study of 19 individuals, 9 of whom had been diagnosed with chronic periodontitis. Five hundred twenty-three operational taxonomic units (OTUs) were obtained from the V1-V2 region and 432 from the V5-V6 region. Key periodontal pathogens like Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia could be identified at the species level with both primer sets. Principal coordinate analysis identified two outliers that were consistently independent of the hypervariable region and method of DNA extraction used. The linear discriminant analysis (LDA) effect size algorithm (LEfSe) identified 80 OTU-level biomarkers of periodontitis and 17 of health. Health- and periodontitis-related clusters of OTUs were identified using a connectivity analysis, and the results confirmed previous studies with several thousands of samples. A machine learning algorithm was developed which was trained on all but one sample and then predicted the diagnosis of the left-out sample (jackknife method). Using a combination of the 10 best biomarkers, 15 of 17 samples were correctly diagnosed. Training the algorithm on time-resolved community profiles might provide a highly sensitive tool to detect the onset of periodontitis.

Degradation of chloroaromatics by Pseudomonas putida GJ31: assembled route for chlorobenzene degradation encoded by clusters on plasmid pKW1 and the chromosome

Pseudomonas putida GJ31 has been reported to grow on chlorobenzene using a meta-cleavage pathway with chlorocatechol 2,3-dioxygenase (CbzE) as a key enzyme. The CbzE-encoding gene was found to be localized on the 180 kb plasmid pKW1 in a cbzTEXGS cluster, which is flanked by transposases and encodes only a partial (chloro)catechol meta-cleavage pathway comprising ferredoxin reductase, chlorocatechol 2,3-dioxygenase, an unknown protein, 2-hydroxymuconic semialdehyde dehydrogenase and glutathione S-transferase. Downstream of cbzTEXGS are located cbzJ, encoding a novel type of 2-hydroxypent-2,4-dienoate hydratase, and a transposon region highly similar to Tn5501. Upstream of cbzTEXGS, traNEOFG transfer genes were found. The search for gene clusters possibly completing the (chloro)catechol metabolic pathway of GJ31 revealed the presence of two additional catabolic gene clusters on pKW1. The mhpRBCDFETP cluster encodes enzymes for the dissimilation of 2,3-dihydroxyphenylpropionate in a novel arrangement characterized by the absence of a gene encoding 3-(3-hydroxyphenyl)propionate monooxygenase and the presence of a GntR-type regulator, whereas the nahINLOMKJ cluster encodes part of the naphthalene metabolic pathway. Transcription studies supported their possible involvement in chlorobenzene degradation. The upper pathway cluster, comprising genes encoding a chlorobenzene dioxygenase and a chlorobenzene dihydrodiol dehydrogenase, was localized on the chromosome. A high level of transcription in response to chlorobenzene revealed it to be crucial for chlorobenzene degradation. The chlorobenzene degradation pathway in strain GJ31 is thus a mosaic encoded by four gene clusters.

Interindividual differences in response to treatment with butyrate-producing Butyricicoccus pullicaecorum 25–3T studied in an in vitro gut model

Butyrate-producing bacteria are promising probiotic candidates to target microbial dysbiosis in gastrointestinal disorders like inflammatory bowel diseases. Butyricicoccus pullicaecorum 25-3(T), a butyrate-producing clostridial cluster IV strain, is such a candidate. Little is known about its abundance in the colon microbiota and its butyrogenic properties. We used the M-SHIME(®), an in vitro simulator for the human intestinal microbial ecosystem, to study the effect of supplementing a single dose of B. pullicaecorum 25-3(T) on lumen- and mucus-associated microbiota of eight individuals. Butyricicoccus pullicaecorum was more abundant in mucus-associated microbiota compared with lumen microbiota. Supplementation with a single dose of B. pullicaecorum 25-3(T) resulted in a temporary increase in B. pullicaecorum bacteria in lumen compartment of all individuals. In two cases, the responders, an increased butyrate production was observed as compared with the control. 16S rRNA gene amplicon sequencing revealed the microbiota of responders to be different as compared to non-responder microbiota. We can conclude that B. pullicaecorum 25-3(T) is a mucus-associated bacterium whose potency to stimulate butyrate production is characterized by a large interindividual variability in terms of composition of the receiving microbial community.

Product Diversity Linked to Substrate Usage in Chain Elongation by Mixed-Culture Fermentation

Acetate and ethanol can be converted to caproic acid by microorganisms through reverse β-oxidation. There is limited insight into the versatility of chain elongation in view of different starting substrates, including even- and odd-carbon carboxylates and alcohols other than ethanol. Thermodynamic analyses show that most elongation pathways are energetically feasible. Through incubations of microbial communities with different substrate-pair combinations, we established that ethanol and propanol were both highly suitable for chain elongation. As an electron acceptor, acetate, propionate, and butyrate readily elongated with ethanol, whereas an adaptation period was necessary for formate. Isobutyrate and longer-chained fatty acids above butyrate were not elongated. The microbial communities converged, and consistent enrichment of Clostridium spp. was observed, independent of the supplied alcohol or carboxylate, with a strain related to Clostridium kluyveri dominating the enrichments. Community analysis also showed phylotypes related to Bacteroidaceae and Microbacteriaceae families in all tests that are capable of converting the base substrates to useful intermediates. These organisms were mainly enriched with methanol or formate. Our overall conclusion is thus that multiple substrates can be used for chain elongation and that this process is carried out by highly similar organisms for direct chain elongation irrespective of the substrate.

Presence does not imply activity: DNA and RNA patterns differ in response to salt perturbation in anaerobic digestion.

BackgroundThe microbial community in anaerobic digestion is mainly monitored by means of DNA-based methods. This may lead to incorrect interpretation of the community parameters, because microbial abundance does not necessarily reflect activity. In this research, the difference between microbial community response on DNA (total community) and RNA (active community) based on the 16S rRNA (gene) with respect to salt concentration and response time was evaluated.ResultsThe application of higher NaCl concentrations resulted in a decrease in methane production. A stronger and faster response to salt concentration was observed on RNA level. This was reflected in terms of microbial community composition and organization, as richness, evenness, and overall diversity were differentially impacted. A higher divergence of community structure was observed on RNA level as well, indicating that total community composition depends on deterministic processes, while the active community is determined by stochastic processes. Methanosaeta was identified as the most abundant methanogen on DNA level, but its relative abundance decreased on RNA level, related to salt perturbation.ConclusionsThis research demonstrated the need for RNA-based community screening to obtain reliable information on actual community parameters and to identify key species that determine process stability.