Construction of Aerobic/Anaerobic-Substrate-Induced Gene Expression Procedure for Exploration of Metagenomes From Subseafloor Sediments

Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

Seasonal and Technological Shifts of the WHO Priority Multi-Resistant Pathogens in Municipal Wastewater Treatment Plant and Its Receiving Surface Water: A Case Study

The present study was focused on the identification of multi-resistant bacteria from the WHO priority pathogens list in the samples taken from different stages of the full-scale municipal wastewater treatment plant and receiving water. Additionally, the seasonal variations of the selected multi-resistant pathogens were analyzed in the samples. In order to the aim of the study, the metagenomic DNA from the collected samples was isolated and sequenced. The samples were collected in three campaigns (spring, summer, autumn). Metagenomic DNA was isolated by the commercial kits, according to the manufacturer’s instruction. Illumina sequencing system was employed, and the R program was used to metagenomic analysis. It was found that the wastewater samples and receiving water contained the multi-resistant bacteria from the WHO priority pathogens list. The seasonal and technological variations affected the distribution of the pathogens in the wastewater. No effect of the effluent on the pathogens in the receiving water was observed. The results indicated that antibiotic-resistant “priority pathogens” from the WHO list are there in the waste- and receiving water. Technological process and seasons effected their distribution in the environment. Metagenomic analysis can be used as sufficient tool in microbiological and human health risk assessment.

Functional screening of a human saliva metagenomic DNA reveal novel resistance genes against sodium hypochlorite and chlorhexidine

Objective Many sections of the health care system are facing a major challenge making infectious disease problematic to treat; antimicrobial resistance (AMR). Identification and surveillance of the resistome have been highlighted as one of the strategies to overcome the problem. This study aimed to screen for AMR genes in an oral microbiota, a complex microbial system continuously exposed to antimicrobial agents commonly used in dental practice. Materials and methods As a significant part of the oral microbiome cannot be conventionally cultured, a functional metagenomic approach was chosen. The human oral metagenomic DNA was extracted from saliva samples collected from 50 healthy volunteers in Norway. The oral metagenomic library was then constructed by ligating partially digested oral metagenome into pSMART BAC vector and introducing into Escherichia coli. The library was screened against antimicrobials in dental practices. All resistant clones were selected and analyzed. Results Screening of the oral metagenomic library against different antimicrobials detected multiple clones with resistance against chlorhexidine, triclosan, erythromycin, tetracycline, and sodium hypochlorite. Bioinformatic analysis revealed both already known resistance genes, including msr, mef(A), tetAB(46), and fabK, and genes that were not previously described to confer resistance, including recA and accB conferring resistance to sodium hypochlorite and chlorhexidine, respectively. Conclusion Multiple clones conferring resistance to antimicrobials commonly used in dental practices were detected, containing known and novel resistant genes by functional-based metagenomics. There is a need for more studies to increase our knowledge in the field.

A metagenomic DNA sequencing assay that is robust against environmental DNA contamination

Metagenomic DNA sequencing is a powerful tool to characterize microbial communities but is sensitive to environmental DNA contamination, in particular when applied to samples with low microbial biomass. Here, we present contamination-free metagenomic DNA sequencing (Coffee-seq), a metagenomic sequencing assay that is robust against environmental contamination. The core idea of Coffee-seq is to tag the DNA in the sample prior to DNA isolation and library preparation with a label that can be recorded by DNA sequencing. Any contaminating DNA that is introduced in the sample after tagging can then be bioinformatically identified and removed. We applied Coffee-seq to screen for infections from microorganisms with low burden in blood and urine, to identify COVID-19 co-infection, to characterize the urinary microbiome, and to identify microbial DNA signatures of inflammatory bowel disease in blood.

Syotti: Scalable Bait Design for DNA Enrichment

Bait-enriched sequencing is a relatively new sequencing protocol that is becoming increasingly ubiquitous as it has been shown to successfully amplify regions of interest in metagenomic samples. In this method, a set of synthetic probes ("baits") are designed, manufactured, and applied to fragmented metagenomic DNA. The probes bind to the fragmented DNA and any unbound DNA is rinsed away, leaving the bound fragments to be amplified for sequencing. This effectively enriches the DNA for which the probes were designed. Most recently, Metsky et al. (Nature Biotech 2019) demonstrated that bait-enrichment is capable of detecting a large number of human viral pathogens within metagenomic samples. In this work, we formalize the problem of designing baits by defining the Minimum Bait Cover problem, which aims to find the smallest possible set of bait sequences that cover every position of a set of reference sequences under an approximate matching model. We show that the problem is NP-hard, and that it remains NP-hard under very restrictive assumptions. This indicates that no polynomial-time exact algorithm exists for the problem, and that the problem is intractable even for small and deceptively simple inputs. In light of this, we design an efficient heuristic that takes advantage of succinct data structures. We refer to our method as Syotti. The running time of Syotti shows linear scaling in practice, running at least an order of magnitude faster than state-of-the-art methods, including the recent method of Metsky et al. At the same time, our method produces bait sets that are smaller than the ones produced by the competing methods, while also leaving fewer positions uncovered. Lastly, we show that Syotti requires only 25 minutes to design baits for a dataset comprised of 3 billion nucleotides from 1000 related bacterial substrains, whereas the method of Metsky et al. shows clearly super-linear running time and fails to process even a subset of 8% of the data in 24 hours. Our implementation is publicly available at https://github.com/jnalanko/syotti.

Probe-mining of endo-1,4-beta-xylanase from goats-rumen bacterial metagenomic DNA data

Endo-1,4-beta-xylanases (xylanases) are classified into 9 glycoside hydrolase families, GH5, 8, 10, 11, 30, 43, 51, 98, and 141 based on the CAZy database. The probe sequences representing the enzymes were constructed from published sequences of actual experimental studies with xylan decomposition activity. From online databases, we found one sequence belonging to the GH5 family, 6 sequences belonging to the GH8 family and 5 sequences belonging to the GH30 family exhibiting xylanase activity. Thus specific probes for xylanase GH8 and GH30 families were designed with the length of 351 and 425 amino acids respectively. The reference values for the probe of the GH8 family were defined as the sequences with maximum score greater than 168, the lowest coverage was 84%, the lowest similarity was 36%; for the probe GH30, the maximum score was greater than 316, the coverage was greater than 98%, the similarity was greater than 41%. Using the built probes, including the probe of the two GH10 and GH11 families, we found 41 xylanase-encoding sequences from the metagenomic DNA data of bacteria in Vietnamese goats’rumen. Of the 41 exploited sequences, 19 were identical to the BGI company's annotation result based on KEGG database, whereas there were 16 sequences that are not annotated by the BGI company. Total 28 of 41 exploited sequences were complete open reading frames, of which the predicted ternary structure was highly similar to the published structures of xylanase.

Differences of Gut Microbiota in the Freshwater Blackworm (Lumbriculus variegatus: Oligochaeta) in Two Different Habitat Conditions

The distribution of organisms is governed by their habitat condition. We analyzed bacterial communities in the gut of the blackworm Lumbriculus variegatus by pyrosequencing of the extracted intestinal metagenomic DNA. Blackworms were collected from two sampling sites with differences in irradiance and riparian vegetation, where site GP7 was covered by riparian vegetation and site GP8 was exposed to sunlight. We obtained the filtered 6414 reads from three samples of each site. At GP7, 271 OTUs were identified, including 32 OTUs unique to the site, whereas at GP8, 238 OTUs were identified, including 22 unique OTUs. Among them, 18 OTUs were shared between both sites. The phylum Proteobacteria was a major component contributing 67.84% and 64.05% of sequences at sites GP7 and GP8, respectively, while each remaining phylum contributed less than 10% at both sites. The two sites differed in microbial community composition and KEGG-indicated biochemical pathways. Community indices such as species richness and Shannon diversity were higher at site GP7 than at GP8. Meanwhile, the abundance of Cyanobacteria was significantly higher at site GP8, while site GP7 showed a greater proportion of genes for membrane transport and carbohydrate metabolism, reflecting differences in food resources.

MINTIA: a metagenomic INserT integrated assembly and annotation tool

The earth harbors trillions of bacterial species adapted to very diverse ecosystems thanks to specific metabolic function acquisition. Most of the genes responsible for these functions belong to uncultured bacteria and are still to be discovered. Functional metagenomics based on activity screening is a classical way to retrieve these genes from microbiomes. This approach is based on the insertion of large metagenomic DNA fragments into a vector and transformation of a host to express heterologous genes. Metagenomic libraries are then screened for activities of interest, and the metagenomic DNA inserts of active clones are extracted to be sequenced and analysed to identify genes that are responsible for the detected activity. Hundreds of metagenomics sequences found using this strategy have already been published in public databases. Here we present the MINTIA software package enabling biologists to easily generate and analyze large metagenomic sequence sets, retrieved after activity-based screening. It filters reads, performs assembly, removes cloning vector, annotates open reading frames and generates user friendly reports as well as files ready for submission to international sequence repositories. The software package can be downloaded from https://github.com/Bios4Biol/MINTIA.

Molecular detection and characterization of bacteria from CSF samples of patients with suspected cerebrospinal meningitis in parts of northern Nigeria using metagenomic DNA extracts

Background: The most commonly used approaches for detection and characterization of bacterial pathogens of meningitis in developing countries include culture, Gram stain, and latex agglutination. The positivity rate of culture is relatively low due to suboptimal storage and transportation conditions, culture practice, and/or antibiotic treatment administered before specimens are collected. Specimens that yield no growth in culture can still be analyzed using molecular methods, and metagenomic DNA (mDNA) extracted directly from clinical samples (CSF) can be used. We aimed to detect and characterize three major bacterial causes of cerebrospinal meningitis (CSM); Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae using mDNA extracted directly from CSF samples. Methodology: Metagenomic DNA templates were prepared directly from CSF specimens collected from 210 patients with suspected CSM. A multiplex Real Time PCR (mRT-PCR) using the ABI StepOne Plus Machine and Taqman Probe chemistry was used in the molecular detection, while serogroup/serotype-specific singleplex RT-PCR was used to characterize all positives samples. Results: Eighty-eight (41.9%) of the 210 samples were positive with the mRT-PCR assay for one or a combination of two of the three bacteria. Of these, 59 (67.1%) were N. meningitidis, 2 (2.3%) were H. influenzae, 3 (3.4%) were S. pneumoniae, 15 (17 %) had co-infections of N. meningitidis with H. influenzae, and 9 (10.2%) had co-infections of H. influenzae and S. pneumoniae. The serogroups of N. meningitidis encountered were A (13.5%), B (23%), C (8.1%), W135 (8.1%), X (5.4%), Y (32.4%), and non-groupable (9.5%). The serotypes of H. influenzae were Hia (3.8%), Hib (57.7%), Hic (3.85%), Hie (11.5%) and Hif (23.1%). The serotypes of S. pneumoniae were Wxy1 (8.3%), Wxy4 (33.3%), Wxy5 (50.0%), and Wxy9 (8.3%). Conclusion: Multiplex RT-PCR is a fast and accurate method for detecting and characterizing serogroups/serotypes of major bacteria implicated in CSM. Isolating DNA directly from CSF improves turnaround time, which will speed up patient care and management. Keywords: Cerebrospinal meningitis, metagenomic DNA, multiplex Real Time PCR, Northern Nigeria French title: Détection moléculaire et caractérisation de bactéries à partir d'échantillons de LCR de patients suspectés de méningite cérébrospinale dans certaines parties du nord du Nigéria à l'aide d'extraits d'ADN métagénomique Contexte: Les approches les plus couramment utilisées pour la détection et la caractérisation des agents pathogènes bactériens de la méningite dans les pays en développement comprennent la culture, la coloration de Gram et l'agglutination au latex. Le taux de positivité de la culture est relativement faible en raison des conditions de stockage et de transport sous-optimales, des pratiques de culture et/ou du traitement antibiotique administré avant le prélèvement des échantillons. Les échantillons qui ne donnent pas de croissance en culture peuvent toujours être analysés à l'aide de méthodes moléculaires, et l'ADN métagénomique (ADNm) extrait directement d'échantillons cliniques (LCR) peut être utilisé. Nous visions à détecter et à caractériser trois causes bactériennes majeures de la méningite cérébrospinale (CSM); Neisseria meningitidis, Haemophilus influenzae et Streptococcus pneumoniae à l'aide d'ADNm extrait directement d'échantillons de LCR. Méthodologie: Des matrices d'ADN métagénomique ont été préparées directement à partir d'échantillons de LCR prélevés sur 210 patients suspects de CSM. Une PCR multiplex en temps réel (mRT-PCR) utilisant la chimie de la machine ABI StepOne Plus et de la sonde Taqman a été utilisée pour la détection moléculaire, tandis que la RT-PCR monoplex spécifique au sérogroupe/sérotype a été utilisée pour caractériser tous les échantillons positifs. Résultats: Quatre-vingt-huit (41,9%) des 210 échantillons étaient positifs avec le test mRT-PCR pour une ou une combinaison de deux des trois bactéries. Parmi ceux-ci, 59 (67,1%) étaient N. meningitidis, 2 (2,3%) étaient H. influenzae, 3 (3,4%) étaient S. pneumoniae, 15 (17%) avaient des co-infections de N. meningitidis avec H. influenzae et 9 (10,2%) avaient des co-infections à H. influenzae et S. pneumoniae. Les sérogroupes de N. meningitidis rencontrés étaient A (13,5%), B (23%), C (8,1%), W135 (8,1%), X (5,4%), Y (32,4%) et non groupables (9,5%). Les sérotypes de H. influenzae étaient Hia (3,8%), Hib (57,7%), Hic (3,85%), Hie (11,5%) et Hif (23,1%). Les sérotypes de S. pneumoniae étaient Wxy1 (8,3%), Wxy4 (33,3%), Wxy5 (50,0%) et Wxy9 (8,3%). Conclusion: La RT-PCR multiplex est une méthode rapide et précise de détection et de caractérisation des sérogroupes/sérotypes des principales bactéries impliquées dans le CSM. Isoler l'ADN directement du LCR améliore le temps de traitement, ce qui accélérera les soins et la gestion des patients. Mots clés: méningite cérébro-spinale, ADN métagénomique, PCR multiplex en temps réel, nord du Nigéria