Microbial dark matter sequences verification in amplicon sequencing and environmental metagenomics data

Although microorganisms constitute the most diverse and abundant life form on Earth, in many environments, the vast majority of them remain uncultured. As it is based on information gleaned mainly from cultivated microorganisms, our current body of knowledge regarding microbial life is partial and does not reflect actual microbial diversity. That diversity is hidden in the uncultured microbial majority, termed by microbiologists as “microbial dark matter” (MDM), a term borrowed from astrophysics. Metagenomic sequencing analysis techniques (both 16S rRNA gene and shotgun sequencing) compare gene sequences to reference databases, each of which represents only a small fraction of the existing microorganisms. Unaligned sequences lead to groups of “unknown microorganisms” that are usually ignored and rarefied from diversity analysis. To address this knowledge gap, we analyzed the 16S rRNA gene sequences of microbial communities from four different environments—a living organism, a desert environment, a natural aquatic environment, and a membrane bioreactor for wastewater treatment. From those datasets, we chose representative sequences of potentially unknown bacteria for additional examination as “microbial dark matter sequences” (MDMS). Sequence existence was validated by specific amplification and re-sequencing. These sequences were screened against databases and aligned to the Genome Taxonomy Database to build a comprehensive phylogenetic tree for additional sequence classification, revealing potentially new candidate phyla and other lineages. These putative MDMS were also screened against metagenome-assembled genomes from the explored environments for additional validation and for taxonomic and metabolic characterizations. This study shows the immense importance of MDMS in environmental metataxonomic analyses of 16S rRNA gene sequences and provides a simple and readily available methodology for the examination of MDM hidden behind amplicon sequencing results

Long-term surveillance of sulfate-reducing bacteria in highly saline industrial wastewater evaporation ponds

Abundance and seasonal dynamics of sulfate-reducing bacteria (SRB), in general, and of extreme halophilic SRB (belonging to Desulfocella halophila) in particular, were examined in highly saline industrial wastewater evaporation ponds over a forty one month period. Industrial wastewater was sampled and the presence of SRB was determined by quantitative real-time PCR (qPCR) with a set of primers designed to amplify the dissimilatory sulfite reductase (dsrA) gene. SRB displayed higher abundance during the summer (106–108 targets ml-1) and lower abundance from the autumn-spring (103–105 targets ml-1). However, addition of concentrated dissolved organic matter into the evaporation ponds during winter immediately resulted in a proliferation of SRB, despite the lower wastewater temperature (12–14°C). These results indicate that the qPCR approach can be used for rapid measurement of SRB to provide valuable information about the abundance of SRB in harsh environments, such as highly saline industrial wastewaters. Low level of H2S has been maintained over five years, which indicates a possible inhibition of SRB activity, following artificial salination (≈16% w/v of NaCl) of wastewater evaporation ponds, despite SRB reproduction being detected by qPCR

Wastewater monitoring of SARS-CoV-2 in on-grid, partially and fully off-grid Bedouin communities in Southern Israel

BackgroundWastewater based epidemiology (WBE) has become an important tool in SARS-CoV-2 surveillance and epidemiology. While WBE measurements generally correlate with observed case numbers in large municipal areas on sewer grids, there are few studies on its utility in communities that are off-grid (non-sewered).Methods and materialsTo explore the applicability of wastewater surveillance in our region, five Bedouin communities along the Hebron Stream in Southern Israel (Negev desert) were sampled. One point (El-Sayed) represents a community with partial connection to the sewer grid system and another point (Um Batin) represents a community with no access to the sewer grid system. The towns of Hura, Lakia, and Tel Al-Sabi/Tel Sheva were on-grid. A total of 87 samples were collected between August 2020 to January 2021 using both grab and composite sampling. RNA was extracted from the raw sewage and concentrated sewage. RT-qPCR was carried out with N1, N2, and N3 gene targets, and findings were compared to human case data from the Israeli Ministry of Health.ResultsSARS-CoV-2 was detected consistently over time in on-grid Bedouin towns (Lakia, Tel Sheva/Tel as-Sabi, and Hura) and inconsistently in smaller, off-grid communities (El-Sayed and Um Batin). The trend in maximum copy number/L appears to be driven by population size. When comparing case numbers normalized to population size, the amount of gene copies/L was inconsistently related to reported case numbers. SARS-CoV-2 was also detected from sewage-impacted environmental waters representing communities with no access to the wastewater grid. When grab sampling and composite sampling data were compared, results were generally comparable however composite sampling produced superior results.ConclusionsThe mismatch observed between detected virus and reported cases could indicate asymptomatic or “silent” community transmission, under-testing within these communities (due to factors like mistrust in government, stigma, misinformation) or a combination therein. While the exact reason for the mismatch between environmental SARS-CoV-2 signals and case numbers remains unresolved, these findings suggest that sewage surveillance, including grab sampling methodologies, can be a critical aspect of outbreak surveillance and control in areas with insufficient human testing and off-grid communities

Pokretljivost bakterija po inertnoj površini posredovana kapilarnim silama

Here we describe an additional type of bacterial migration in which bacterial cells migrate vertically across a non-nutritive solid surface carried by capillary forces. Unlike standard motility experiments, these were run on a glass slide inserted into a Falcon tube, partly immersed in a nutrient medium and partly exposed to air. Observations revealed that capillary forces initiated upward cell migration when biofilm was formed at the border between liquid and air. The movement was facilitated by the production of extracellular polymeric substances (EPS). This motility differs from earlier described swarming, twitching, gliding, sliding, or surfing, although these types of movements are not excluded. We therefore propose to call it “capillary movement of biofilm”. This phenomenon may be an ecologically important mode of bacterial motility on solid surfaces.Primijećen je i prikazan dosad neopisan način pokretljivosti bakterija u obliku biofilma. Pokazano je kako se bakterijske stanice iz biofilma formiranoga na razmeđi tekućine i zraka gibaju okomito po staklenom predmetnom stakalcu, inertnoj, nehranjivoj ali djelomično vlažnoj površini. Takva površina, izložena zraku, uvelike se razlikuje od površine hranjivog agara, ili od površine prekrivene hranjivim agarom, kakve se koriste u standardnim testovima za ispitivanje bakterijske pokretljivosti u biofilmu. Sudeći prema opažanjima, stanice se prvotno gibaju posredstvom kapilarnih sila, a kada se formira biofilm, stvaranje izvanstanične polimerne tvari (EPS) potiče daljnju migraciju bakterija. Takva pokretljivost opažena je kod svih testiranih a fiziološki bitno različitih bakterijskih vrsta. Kako se eksperimentalni postav značajno razlikuje od standardnih testova praćenja bakterijske pokretljivosti, kao što su rojenje (swarming), trzanje (twitching) ili klizanje po površini (gliding, sliding ili surfing), predložen je generički naziv “kapilarna pokretljivost biofilma”. Ovdje opisani fenomen mogao bi biti značajan ekološki čimbenik pokretljivosti bakterija u okolišu

Highly sensitive and specific detection of E. coli by a SERS nanobiosensor chip utilizing metallic nanosculptured thin films

A nanobiosensor chip, utilizing surface enhanced Raman spectroscopy (SERS) on nanosculptured thin films (nSTFs) of silver, was shown to detect Escherichia coli (E. coli) bacteria down to the concentration level of a single bacterium. The sensor utilizes highly enhanced plasmonic nSTFs of silver on a silicon platform for the enhancement of Raman bands as checked with adsorbed 4-aminothiophenol molecules. T-4 bacteriophages were immobilized on the aforementioned surface of the chip for the specific capture of target E. coli bacteria. To demonstrate that no significant non-specific immobilization of other bacteria occurs, three different, additional bacterial strains, Chromobacterium violaceum, Paracoccus denitrificans and Pseudomonas aeruginosa were used. Furthermore, experiments performed on an additional strain of E. coli to address the specificity and reusability of the sensor showed that the sensor operates for different strains of E. coli and is reusable. Time resolved phase contrast microscopy of the E. coli-T4 bacteriophage chip was performed to study its interaction with bacteria over time. Results showed that the present sensor performs a fast, accurate and stable detection of E. coli with ultra-small concentrations of bacteria down to the level of a single bacterium in 10 μl volume of the sample

Heat-stress and light-stress induce different cellular pathologies in the symbiotic dinoflagellate during coral bleaching

Coral bleaching is a significant contributor to the worldwide degradation of coral reefs and is indicative of the termination of symbiosis between the coral host and its symbiotic algae (dinoflagellate; Symbiodinium sp. complex), usually by expulsion or xenophagy (symbiophagy) of its dinoflagellates. Herein, we provide evidence that during the earliest stages of environmentally induced bleaching, heat stress and light stress generate distinctly different pathomorphological changes in the chloroplasts, while a combined heat- and light-stress exposure induces both pathomorphologies; suggesting that these stressors act on the dinoflagellate by different mechanisms. Within the first 48 hours of a heat stress (32°C) under low-light conditions, heat stress induced decomposition of thylakoid structures before observation of extensive oxidative damage; thus it is the disorganization of the thylakoids that creates the conditions allowing photo-oxidative-stress. Conversely, during the first 48 hours of a light stress (2007 µmoles m−2 s−1 PAR) at 25°C, condensation or fusion of multiple thylakoid lamellae occurred coincidently with levels of oxidative damage products, implying that photo-oxidative stress causes the structural membrane damage within the chloroplasts. Exposure to combined heat- and light-stresses induced both pathomorphologies, confirming that these stressors acted on the dinoflagellate via different mechanisms. Within 72 hours of exposure to heat and/or light stresses, homeostatic processes (e.g., heat-shock protein and anti-oxidant enzyme response) were evident in the remaining intact dinoflagellates, regardless of the initiating stressor. Understanding the sequence of events during bleaching when triggered by different environmental stressors is important for predicting both severity and consequences of coral bleachin

‘Next-base’ effect on PCR amplification

The base adjacent to the 3′ end of universal PCR primers targeting the 16S rRNA gene is often variable and apparently biases the microbial community composition as represented by PCR-based surveys. To test this hypothesis, four templates of 44 bases each and two complementary primers (21 bases) were designed to differ only in the bases adjacent to the primers, and their amplification efficiencies were evaluated using quantitative PCR. For extension temperatures of 72°C, 73°C and 74°C, improvement in initial amplification efficiency was observed for templates with guanine or cytosine at the position contiguous to the primers. However, no clear preference was observed when extension temperature was lowered to 70°C. Shortening the primers by one base, so that the variable position was located two base pairs downstream from the primer, attenuated but did not eliminate this bias. A conformational change of the quaternary polymerase – primer – template – dNTP complex upon commencing of polymerization is thought to be a rate-limiting step. A possible explanation for the observed bias is the stabilization of this complex by the adjacent guanine or cytosine. Reducing PCR extension temperature to 70°C minimizes amplification biases caused by variable template-contiguous bases to the 3′ end of universal PCR primers. Next-base nucleotide composition should be taken in consideration in designing primers targeting 16S rRNA or other functional genes used in microbial ecology studies

Genome Analysis of a Novel Broad Host Range Proteobacteria Phage Isolated from a Bioreactor Treating Industrial Wastewater

Bacteriophages are viruses that infect bacteria, and consequently they have a major impact on the development of a microbial population. In this study, the genome of a novel broad host range bacteriophage, Aquamicrobium phage P14, isolated from a wastewater treatment plant, was analyzed. The Aquamicrobium phage P14 was found to infect members of different Proteobacteria classes (Alphaproteobacteria and Betaproteobacteria). This phage contains a 40,551 bp long genome and 60% of its genes had blastx hits. Furthermore, the bacteriophage was found to share more than 50% of its genes with several podoviruses and has the same gene order as other polyvalent bacteriophages. The results obtained in this study led to the conclusion that indeed general features of the genome of the Aquamicrobium phage P14 are shared with other broad host range bacteriophages, however further analysis of the genome is needed in order to identify the specific mechanisms which enable the bacteriophage to infect both Alphaproteobacteria and Betaproteobacteria

Application of a unique miniature MBR for screening the biodegradation of brominated flame retardants

A unique miniature membrane bioreactor (mMBR) was designed and applied to examine the biodegradability of two complex organic compounds belonging to a family of brominated flame retardants (BFR) under continuous culture conditions using a bacterial consortium. BFRs are a widely used group of anthropogenic environmental contaminants. Many of these compounds are toxic, persistent, have limited biodegradability, and tend to bioaccumulate in the environment. Their widespread production and use combined with the inappropriate treatment and disposal of industrial wastewater have caused myriad global health and environmental concerns. Dibromoneopentyl glycol (DBNPG) and tribromoneopentyl alcohol (TBNPA) are aliphatic BFRs, classified as recalcitrant compounds, having half-lives of more than 100 years. Following successful debromination and complete biodegradation of the two target compounds in the mMBR, we used molecular and bioinformatic techniques to track changes in bacterial community composition during the biodegradation process.Published versio