City-scale monitoring of antibiotic resistance genes by digital PCR and metagenomics

Background Anthropogenic activities significantly contribute to the dissemination of antibiotic resistance genes (ARGs), posing a substantial threat to humankind. The development of methods that allow robust ARG surveillance is a long-standing challenge. Here, we use city-scale monitoring of ARGs by using two of the most promising cutting-edge technologies, digital PCR (dPCR) and metagenomics. Methods ARG hot-spots were sampled from the urban water and wastewater distribution systems. Metagenomics was used to provide a broad view of ARG relative abundance and richness in the prokaryotic and viral fractions. From the city-core ARGs in all samples, the worldwide dispersed sul2 and tetW conferring resistance to sulfonamide and tetracycline, respectively, were monitored by dPCR and metagenomics. Results The largest relative overall ARG abundance and richness were detected in the hospital wastewater and the WWTP inlet (up to ≈6,000 ARGs/Gb metagenome) with a large fraction of unclassified resistant bacteria. The abundance of ARGs in DNA and RNA contigs classified as viruses was notably lower, demonstrating a reduction of up to three orders of magnitude compared to contigs associated to prokaryotes. By metagenomics and dPCR, a similar abundance tendency of sul2 and tetW was obtained, with higher abundances in hospital wastewater and WWTP input (≈125–225 ARGs/Gb metagenome). dPCR absolute abundances were between 6,000 and 18,600 copies per ng of sewage DNA (≈105–7 copies/mL) and 6.8 copies/mL in seawater near the WWTP discharging point. Conclusions dPCR was more sensitive and accurate, while metagenomics provided broader coverage of ARG detection. While desirable, a reliable correlation of dPCR absolute abundance units into metagenomic relative abundance units was not obtained here (r2 < 0.4) suggesting methodological factors that introduce variability. Evolutionary pressure does not significantly select the targeted ARGs in natural aquatic environments.This project was supported with UAIND18-05 grant from the Program “Predoctoral Training in Collaboration with Companies” by the Office of the Vice President for Research, Development, and Innovation (University of Alicante). Funds were also provided by the Hospital Elche Crevillente Salud SL (ref. HOSPITALECLHE1-18Y)

Novel RNA viruses from the Atlantic Ocean: Ecogenomics, biogeography, and total virioplankton mass contribution from surface to the deep ocean

Marine viruses play a major role in the energy and nutrient cycle and affect the evolution of their hosts. Despite their importance, there is still little knowledge about RNA viruses. Here, we have explored the Atlantic Ocean, from surface to deep (4.296 m), and used viromics and quantitative methods to unveil the genomics, biogeography, and the mass contribution of RNA viruses to the total viroplankton. A total of 2481 putative RNA viral contigs (>500 bp) and 107 larger bona fide RNA viral genomes (>2.5 kb) were identified; 88 of them representing novel viruses belonging mostly to two clades: Yangshan assemblage (sister clade to the class Alsuviricetes) and Nodaviridae. These viruses were highly endemic and locally abundant, with little or no presence in other oceans since only ≈15% of them were found in at least one of the Tara sampling metatranscriptomes. Quantitative data indicated that the abundance of RNA viruses in the surface and deep chlorophyll maximum zone was within ≈106 VLP/mL representing a potential contribution of 5.2%–24.4% to the total viroplankton community (DNA and RNA viruses), with DNA viruses being the predominant members (≈107 VLP/mL). However, for the deep sample, the observed trend was the opposite, although as further discussed, several biases should be considered. Together these results contribute to our understanding of the diversity, abundance, and distribution of RNA viruses in the oceans and provide a basis for further investigation into their ecological roles and biogeography.This work was supported by the Generalitat Valenciana ACIF2020 grant and by the research grants funded by Spanish Ministry of Science and Innovation (refs. RTI2018-094248-B-I00 and PID2021-125175OB-I00), and by the Gordon and Moore Foundation (ref. 5334)

Retinitis pigmentosa is associated with shifts in the gut microbiome

The gut microbiome is known to influence the pathogenesis and progression of neurodegenerative diseases. However, there has been relatively little focus upon the implications of the gut microbiome in retinal diseases such as retinitis pigmentosa (RP). Here, we investigated changes in gut microbiome composition linked to RP, by assessing both retinal degeneration and gut microbiome in the rd10 mouse model of RP as compared to control C57BL/6J mice. In rd10 mice, retinal responsiveness to flashlight stimuli and visual acuity were deteriorated with respect to observed in age-matched control mice. This functional decline in dystrophic animals was accompanied by photoreceptor loss, morphologic anomalies in photoreceptor cells and retinal reactive gliosis. Furthermore, 16S rRNA gene amplicon sequencing data showed a microbial gut dysbiosis with differences in alpha and beta diversity at the genera, species and amplicon sequence variants (ASV) levels between dystrophic and control mice. Remarkably, four fairly common ASV in healthy gut microbiome belonging to Rikenella spp., Muribaculaceace spp., Prevotellaceae UCG-001 spp., and Bacilli spp. were absent in the gut microbiome of retinal disease mice, while Bacteroides caecimuris was significantly enriched in mice with RP. The results indicate that retinal degenerative changes in RP are linked to relevant gut microbiome changes. The findings suggest that microbiome shifting could be considered as potential biomarker and therapeutic target for retinal degenerative diseases.This study was funded by the Spanish Ministry of Economy Industry and Competitiveness (MINECO-FEDER BFU2015-67139-R and RTI2018-094248-B-I00), Spanish Ministry of Science and Innovation (MICINN-FEDER PID2019-106230RB-I00), Instituto de Salud Carlos III co-financed by European Regional Development funds (RETICS-FEDER RD16/0008/0016), Asociación Retina Asturias (ASOCIACIONRETINA1-20I), FARPE-FUNDALUCE (FUNDALUCE18-01), Generalitat Valenciana (FEDER IDIFEDER/2017/064) and Alicante’s University (UAIND18-05A)

High-Throughput 16S rRNA Sequencing to Assess Potentially Active Bacteria and Foodborne Pathogens: A Case Example in Ready-to-Eat Food

Technologies to detect the entire bacterial diversity spectra and foodborne pathogens in food represent a fundamental advantage in the control of foodborne illness. Here, we applied high-throughput 16S rRNA sequencing of amplicons obtained by PCR and RT-PCR from extracted DNA and RNA targeting the entire bacterial community and the active bacterial fraction present in some of the most consumed and distributed ready-to-eat (RTE) salad brands in Europe. Customer demands for RTE food are increasing worldwide along with the number of associated foodborne illness and outbreaks. The total aerobic bacterial count in the analyzed samples was in the range of 2–4 × 106 CFU/g (SD ± 1.54 × 106). Culture validated methods did not detect Salmonella spp., Escherichia coli, and other fecal coliforms. 16S rRNA gene Illumina next-generation sequencing (NGS) data were congruent with these culture-based results and confirmed that these and other well-known foodborne bacterial pathogens, such as Listeria, were not detected. However, the fine-resolution of the NGS method unveiled the presence of the opportunistic pathogens Aeromonas hydrophyla and Rahnella aquatilis (relative frequency of 1.33–7.33%) that were metabolically active in addition to non-pathogenic, active members of Yersinia spp. (relative frequency of 0.0015–0.003%). The common ail and foxA marker genes of Yersinia enterocolitica were not detected by qPCR. Finally, our NGS data identified to non-pathogenic Pseudomonas spp. as the most abundant and metabolically active bacteria in the analyzed RTE salads (53–75% of bacterial abundance). Our data demonstrate the power of sequencing, in parallel, both 16S rRNA and rDNA to identify and discriminate those potentially and metabolically active bacteria and pathogens to provide a more complete view that facilitates the control of foodborne diseases, although further work should be conducted to determine the sensitivity of this method for targeting bacteria.This work was supported by the Spanish Ministry of Economy and Competitiveness (ref. RTI2018-094248-B-I00) and the Gordon and Betty Moore Foundation (grant 5334)

Droplet Digital PCR for Estimating Absolute Abundances of Widespread Pelagibacter Viruses

Absolute abundances of prokaryotes are typically determined by FISH. Due to the lack of a universal conserved gene among all viruses, metagenomic fragment recruitment is commonly used to estimate the relative viral abundance. However, the paucity of absolute virus abundance data hinders our ability to fully understand how viruses drive global microbial populations. The cosmopolitan marine Pelagibacter ubique is host for the highly widespread HTVC010P pelagiphage isolate and the extremely abundant uncultured virus vSAG 37-F6 recently discovered by single-virus genomics. Here we applied droplet digital PCR (ddPCR) to calculate the absolute abundance of these pelagiphage genotypes in the Mediterranean Sea and the Gulf of Maine. Abundances were between 360 and 8,510 virus mL-1 and 1,270–14,400 virus mL-1 for vSAG 37-F6 and HTVC010P, respectively. Illumina PCR-amplicon sequencing corroborated the absence of ddPCR non-specific amplifications for vSAG 37-F6, but showed an overestimation of 6% for HTVC010P from off-targets, genetically unrelated viruses. Absolute abundances of both pelagiphages, two of the most abundance marine viruses, suggest a large viral pelagiphage diversity in marine environments, and show the efficiency and power of ddPCR to disentangle the structure of marine viral communities. Results also highlight the need for a standardized workflow to obtain accurate quantification that allows cross data comparison.This work has been supported by Spanish Ministry of Economy and Competitiveness (Ref. RTI2018-094248-B-I00), Generalitat Valenciana (Ref. ACOM/2015/133 and ACIF/2015/332), and Gordon and Betty Moore Foundation (Grant 5334)

Historia de un viaje: dinámica de los genes de resistencia a antibióticos, del cuerpo humano al ecosistema

La resistencia a antimicrobianos es un problema a escala global que ha sido reconocido como una de las 10 principales amenazas para la salud pública mundial por parte de la Organización Mundial de la Salud. Dada su gran importancia, el análisis de los genes de resistencia a antibióticos, que otorgan resistencia frente a antibióticos a las bacterias que los tienen, pasa a tener un papel crucial para el desarrollo de estrategias que reduzcan la resistencia a antimicrobianos existente. Sabemos de la presencia de genes de resistencia a antibiótico está muy extendida en diferentes ambientes, y en esta tesis, centramos esfuerzos analizando puntos calientes y posibles vías de dispersión de GRA como son el aire y el agua. Para ello se aplicará principalmente la metagenómica, una de las metodologías no basadas en cultivo que mayor información puede dar sobre una muestra dada. Los puntos calientes de genes de resistencia a antibióticos analizados en este trabajo son el ser humano, aguas de depuradora y otras aguas relevantes y el aire de hospitales y depuradoras, este último medio, a pesar de su importancia en la dispersión de genes de resistencia a antibiótico ha sido menos explorado por sus dificultades técnicas y falta de estandarización. Además de puntos calientes de dispersión de genes de resistencia a antibiótico, también analizamos la dispersión desde éstos a ambientes prístinos, en los que la influencia del hombre es inexistente o escasa.Esta tesis ha sido financiada gracias a la ayuda para la realización de contratos destinados a la formación predoctoral en colaboración con empresas (UAIND) con número de referencia I-PI 95-18 en colaboración con el Vicerrectorado de Investigación y Transferencia de Conocimiento de la Universidad de Alicante y el Hospital Universitario del Vinalopó

A Resistome Roadmap: From the Human Body to Pristine Environments

A comprehensive characterization of the human body resistome [sets of antibiotic resistance genes (ARGs)] is yet to be done and paramount for addressing the antibiotic microbial resistance threat. Here, we study the resistome of 771 samples from five major body parts (skin, nares, vagina, gut, and oral cavity) of healthy subjects from the Human Microbiome Project (HMP) and addressed the potential dispersion of ARGs in pristine environments. A total of 28,714 ARGs belonging to 235 different ARG types were found in the HMP proteome dataset (n = 9.1 × 107 proteins analyzed). Our study reveals a distinct resistome profile (ARG type and abundance) between body sites and high interindividual variability. Nares had the highest ARG load (≈5.4 genes/genome) followed by the oral cavity, whereas the gut showed one of the highest ARG richness (shared with nares) but the lowest abundance (≈1.3 genes/genome). The fluroquinolone resistance genes were the most abundant in the human body, followed by macrolide–lincosamide–streptogramin (MLS) or tetracycline. Most ARGs belonged to common bacterial commensals and multidrug resistance trait were predominant in the nares and vagina. Many ARGs detected here were considered as low risk for human health, whereas only a few of them, such as BlaZ, dfrA14, dfrA17, or tetM, were classified as high-risk ARG. Our data also provide hope, since the spread of common ARG from the human body to pristine environments (n = 271 samples; 77 Gb of sequencing data and 2.1 × 108 proteins analyzed) thus far remains very unlikely (only one case found in an autochthonous bacterium from a pristine environment). These findings broaden our understanding of ARG in the context of the human microbiome and the One-Health Initiative of WHO uniting human host–microbes and environments as a whole.This project was supported by the Office of the Vice President for Research, Development, and Innovation (University of Alicante) with a grant from the Program “Predoctoral Training in Collaboration with Companies” (ref. UAIND18-05). Funds were also provided by the Hospital Elche Crevillente Salud SL (ref. HOSPITALECLHE1-18Y)

Insights into the antibiotic resistance dissemination in a wastewater effluent microbiome: bacteria, viruses and vesicles matter

Wastewater treatment plants effluents are considered as hotspots for the dispersion of antibiotic resistance genes (ARGs) into natural ecosystems. The bacterial resistome (ARG collection in a metagenome) analyses have provided clues on antibacterial resistance dynamics. However, viruses and vesicles are frequently ignored. Here, we addressed the bacterial, viral and vesicle resistomes from a representative wastewater effluent in natural conditions and amended with polymyxin, which is used as a last resort antibiotic. Metagenomics showed that the natural prokaryotic resistome was vast (9000 ARG hits/Gb metagenome) and diverse, while viral resistome was two orders of magnitude lower (50 ARG hits/Gb metagenome) suggesting that viruses rarely encoded ARGs. After polymyxin amendment, data showed no ARG enrichment – including to polymyxin – in the microbiome. Remarkably, microbiomes responded to polymyxin with a vast release of putative vesicles (threefold increase compared with the control), which might be used as 'traps' to decrease the antibiotic concentration. Intriguingly, although polymyxin resistance genes (PRGs) were rare in the microbiome (0.018% of total ARG found), in the viral and vesicle fractions, PRGs were more abundant (0.5%–0.8% of total ARG found). Our data suggest that vesicles could have a more active role in the context of transmission of antibiotic resistances.This work has been supported by Ministerio de Ciencia, Innovación y Universidades (ref. RTI2018-094248-B-I00), Generalitat Valenciana (refs. ACOM/2015/133 and ACIF/2015/332), and Gordon and Betty Moore Foundation (grant 5334)

Minimal-moderate variation of human oral virome and microbiome in IgA deficiency

Immunoglobulin A (IgA) is the dominant antibody found in our mucosal secretions and has long been recognized to play an important role in protecting our epithelium from pathogens. Recently, IgA has been shown to be involved in gut homeostatic regulation by ‘recognizing’ and shaping our commensal microbes. Paradoxically, yet selective IgA-deficiency is often described as asymptomatic and there is a paucity of studies only focused on the mice and human gut microbiome context fully ignoring other niches of our body and our commensal viruses. Here, we used as a model the human oral cavity and employed a holistic view and studied the impact of IgA deficiency and also common variable IgA and IgM immunodeficiencies (CVID), on both the human virome and microbiome. Unexpectedly, metagenomic and experimental data in human IgA deficiency and CVID indicate minimal-moderate changes in microbiome and virome composition compared to healthy control group and point out to a rather functional, resilient oral commensal viruses and microbes. However, a significant depletion (two fold) of bacterial cells (p-value < 0.01) and viruses was observed in IgA-deficiency. Our results demonstrate that, within the limits of our cohort, IgA role is not critical for maintaining a rather functional salivary microbiome and suggest that IgA is not a major influence on the composition of abundant commensal microbes.This work has been supported by Spanish Ministry of Economy and Competitiveness (refs. CGL2013-40564-R, RTI2018-094248-B-I00, and SAF2013-49267-EXP)

Short-term high-fat feeding exacerbates degeneration in retinitis pigmentosa by promoting retinal oxidative stress and inflammation

A high-fat diet (HFD) can induce hyperglycemia and metabolic syndromes that, in turn, can trigger visual impairment. To evaluate the acute effects of HFD feeding on retinal degeneration, we assessed retinal function and morphology, inflammatory state, oxidative stress, and gut microbiome in dystrophic retinal degeneration 10 (rd10) mice, a model of retinitis pigmentosa, fed an HFD for 2 to 3 wk. Short-term HFD feeding impaired retinal responsiveness and visual acuity and enhanced photoreceptor degeneration, microglial cell activation, and Müller cell gliosis. HFD consumption also triggered the expression of inflammatory and oxidative markers in rd10 retinas. Finally, an HFD caused gut microbiome dysbiosis, increasing the abundance of potentially proinflammatory bacteria. Thus, HFD feeding drives the pathological processes of retinal degeneration by promoting oxidative stress and activating inflammatory-related pathways. Our findings suggest that consumption of an HFD could accelerate the progression of the disease in patients with retinal degenerative disorders.This work was supported by grants from the Spanish Ministry of the Economy and Competitiveness (RTI2018-094248-B-I00), Spanish Ministry of Science and Innovation cofinanced by European Regional Development Fund (MICINN-FEDER PID2019-106230RB-I00), Instituto de Salud Carlos III co-financed by European Regional Development Fund (RETICS-FEDER-RD16/0008/0016), Asociación Retina Asturias (ASOCIACIONRETINA1-20I), Federación de Asociaciones de Retinosis Pigmentaria de España and Fundación Lucha Contra la Ceguera (FUNDALUCE18-01), and Generalitat Valenciana (PROMETEO/2021/024, IDIFEDER/2017/064)