Phylogenetic characterization and quantification of ammonia-oxidizing archaea and bacteria from Lake Kivu in a long-term microcosm incubation

A microcosm cultivation-based method was set up to investigate the growth of ammonia-oxidizing archaea (AOA), isolated from a water sample acquired at a depth of 50 m from the northern basin of Lake Kivu. For this purpose, both CARD-FISH and qPCR targeting of archaeal 16S rRNA and amoA genes were used. Archaeal cell growth at the end of the 246-day microcosm experiment accounted for 35 % of the SybrGold-stained cells, which corresponded to 6.61 × 106 cells/ml and 1.76 ± 0.09 × 106 archaeal 16S rRNA gene copies/ml. Clone libraries and DGGE fingerprinting confirmed the dominance of AOA phylotypes in the archaeal community microcosm. The majority of the identified archaeal 16S rRNA gene sequences in the clone libraries were affiliated with Thaumarchaeota Marine Group 1.1a. Subsequent cultivation of the AOA community on deep-well microtiter plates in medium containing different carbon sources to stimulate archaeal growth failed to show significant differences in archaeal abundance (ANOVA t14 = –1.058, P = 0.308 and ANOVA t14 = 1.584, P = 0.135 for yeast extract and simple organic acids, respectively). The lack of growth stimulation by organic compounds is in concordance with the oligotrophic status of Lake Kivu. Finally, the addition of antibiotics to the growth medium resulted in archaeal cell counts that were significantly lower than those obtained from cultures in antibiotic-free medium (ANOVA t14 = 12.12, P < 0.001). [Int Microbiol 2013; 16(3):177-189]Keywords: ammonia-oxidizing archaea and bacteria · ammonia monooxygenase alpha subunit (amoA) · Lake Kivu ·microcosm · multi-color CARD-FIS

The molar extinction coefficient of bacteriochlorophyll e and the pigment stoichiometry in Chlorobium phaeobacteroides

We have determined the molar extinction coefficient of bacteriochlorophyll (BChl) e, the main light-harvesting pigment from brown-coloured photosynthetic sulfur bacteria. The extinction coefficient was determined using pure[Pr,E]BChl eF isolated by reversed-phase HPLC from crude pigment extracts of Chlorobium (Chl.) phaeobacteroides strain CL1401. The extinction coefficients at the Soret and Qy bands were determined in four organic solvents. The extinction coefficient of BChl e differs from those of other related Chlorobium chlorophylls (BChl c and BChl d) but is similar to that of chlorophyll b. The determined extinction coefficient was used to calculate the stoichiometric BChl e to BChl a and BChl e to carotenoids ratios in whole cells and isolated chlorosomes from Chl. phaeobacteroides strain CL1401 using the spectrum-reconstruction method (SRCM) described by Naqvi et al. (1997) (Spectrochim Acta A Mol Biomol Spectrosc 53: 2229–2234). In isolated chlorosomes, BChl a content was ca. 1% of the total BChl content and the stoichiometric ratio of BChl e to carotenoids was 6. In whole cells,however, BChl a content was 3–4%, owing to the presence of BChl a-containing elements, i.e. FMO protein and reaction centre. An average of 5 BChl e molecules per carotenoid was determined in whole cells.EU(Contract No FMRX–CT96–0081). Ministerio de Educación y Ciencia (Ref. BIO96–1229–002–01)Peer reviewe

Nanosecond laser photolysis studies of chlorosomes and artificial aggregates containing bacteriochlorophyll e: Evidence for the proximity of carotenoids and bacteriochlorophyll a in chlorosomes from Chlorobium phaeobacteroides strain CL1401

Time-resolved, laser-induced changes in absorbance, ΔA(λ; t), have been recorded with a view to probing pigment–pigment interactions in chlorosomes (control as well as carotenoid-depleted) and artificial aggregates of bacteriochlorophyll e (BChle). Control chlorosomes were isolated from Chlorobium phaeobacteroides strain CL1401, whose chromophores comprise BChle, bacteriochlorophyll a (BChla) and several carotenoid (Car) pigments; Car-depleted chlorosomes, from cells grown in cultures containing 2-hydroxybiphenyl. Artificial aggregates were prepared by dispersing BChle in aqueous phase in the presence of monogalactosyl diglyceride. In chlorosomes ΔA(λ; t) shows, besides a signal attributable to triplet Car (with a half-life of about 4 μs), signals in the Qy regions of both BChl. The BChla signal decays at the same rate as the Car signal, which is explained by postulating that some Car are in intimate contact with some baseplate BChla pigments, and that when a ground-state Car changes into a triplet Car, the absorption spectrum of its BChla neighbors undergoes a concomitant change (termed transient environment-induced perturbation). The signal in the Qy-region of BChle behaves differently: its amplitude falls, under reducing conditions, by more than a factor of two during the first 0.5 μs (a period during which the Car signal suffers negligible diminution), and is much smaller under nonreducing conditions. The BChle signal is also attributed to transient environment-induced perturbation, but in this case the perturber is a BChle photoproduct (probably a triplet or a radical ion). The absence of long-lived BChle triplets in all three systems, and of long-lived BChla triplets in chlorosomes, indicates that BChle in densely packed assemblies is less vulnerable to photodamage than monomeric BChle and that, in chlorosome, BChla rather than BChle needs, and receives, photoprotection from an adjacent Car.Research Council of Norway. EU (contract FMRX-CT96-0081)Peer reviewe

Surveilling the SARS -CoV-2 in sewage: the catalan case

Introduction. Shortly after the outbreak of the COVID-19 pandemic, scientists renewed their interest on the application of wastewater-based epidemiology (WBE) to track the communal circulation of SARS-CoV-2 through the quantification of its genetic traces in sewage. At national scale, such strategy was firstly implemented in The Netherlands in February 2020 and, following the Dutch example, similar sewage surveillance programs were later put into action by other countries at different scales and coverages. At the time of writing, 58 countries are currently monitoring the circulation of SARS-CoV-2 in wastewater as reported at the COVID-Poops19 website. The purpose of this work is to describe the roadmap for the implementation of a wastewater surveillance network at a national scale and to discuss the main challenges faced during its functioning. Material and methods. It should be noted that all data generated are free for scientific use, and it can be downloaded from a public repository at the Zenodo website. The database is weekly updated and contains all molecular data obtained from the samples analyzed. Results. In Catalonia, a Spanish region of 7,5 million inhabitants, the Public Health Agency of the Catalan government and the Catalan Water Agency promoted and funded the implementation of the Catalan Surveillance Network of SARSCoV-2 in Sewage (SARSAIGUA) to provide information on the circulation of SARSCoV-2 at community level that complement epidemiological & clinical data. SARSAIGUA started in 2020 by monitoring 56 WWTPs that assist 193 municipalities, representing 80% of the Catalan population. Within less than 72 hours, weekly samples are collected, analyzed, and results reported to Health authorities and finally published in an on-line dashboard. After 19 months of monitoring, the normalized daily loads of SARS-CoV-2 genes in the 56 WWTPs monitored, fairly matched the sum of COVID-19 cases along the successive pandemic waves. Moreover, a good fit was obtained between the aggregated viral load (gen copies/day/100.000 inhabitants) and the epidemiological evolution of diagnosed cases in the municipalities, served by the monitored WWTPs (rxy=0.59). In 2021, SARSAIGUA started the monitoring of SARS-CoV-2 variants by sequencing sewage samples every two weeks using Oxford nanopore technology and ARCTIC primers targeting the S gene. The deployment of this sequencing approach has allowed to track the introduction and spread of the Omicron variant and the concomitant wane of the Delta variant across the territory. Conclusions. Overall, and despite the difficulties and limitations associated to the inherent complexity of wastewater, the usefulness of WBE to rapidly detect viral transmission at community level is very helpful to Health authorities to better manage the pandemic situation. This is particularly relevant under the current scenario, where new emerging SARS-CoV-2 variants with higher fitness and transmission potential outcompete old ones in a weekly time scale. Acknowledgment. The research was realised in the JPIAMR projects: (PhageLand) – 22.80013.8007.1

Persistence of low pathogenic avian influenza virus in artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate

Altres ajuts: acords transformatius de la UABInstituto Nacional de Investigación y Tecnologı́a Agraria y Alimentaria PID2020-114060RR-C33-INFLUOMAAvian influenza viruses (AIVs) can affect wildlife, poultry, and humans, so a One Health perspective is needed to optimize mitigation strategies. Migratory waterfowl globally spread AIVs over long distances. Therefore, the study of AIV persistence in waterfowl staging and breeding areas is key to understanding their transmission dynamics and optimizing management strategies. Here, we used artificial streams mimicking natural conditions of waterfowl habitats in the Mediterranean climate (day/night cycles of photosynthetic active radiation and temperature, low water velocity, and similar microbiome to lowland rivers and stagnant water bodies) and then manipulated temperature and sediment presence (i.e., 10-13 °C vs. 16-18 °C, and presence vs. absence of sediments). An H1N1 low pathogenic AIV (LPAIV) strain was spiked in the streams, and water and sediment samples were collected at different time points until 14 days post-spike to quantify viral RNA and detect infectious particles. Viral RNA was detected until the end of the experiment in both water and sediment samples. In water samples, we observed a significant combined effect of temperature and sediments in viral decay, with higher viral genome loads in colder streams without sediments. In sediment samples, we didn't observe any significant effect of temperature. In contrast to prior laboratory-controlled studies that detect longer persistence times, infectious H1N1 LPAIV was isolated in water samples till 2 days post-spike, and none beyond. Infectious H1N1 LPAIV wasn't isolated from any sediment sample. Our results suggest that slow flowing freshwater surface waters may provide conditions facilitating bird-to-bird transmission for a short period when water temperature are between 10 and 18 °C, though persistence for extended periods (e.g., weeks or months) may be less likely. We hypothesize that experiments simulating real environments, like the one described here, provide a more realistic approach for assessing environmental persistence of AIVs

Dynamics of SARS-CoV-2 Alpha (B.1.1.7) variant spread: The wastewater surveillance approach

Wastewater based epidemiology (WBE) offers an overview of the SARS-CoV-2 variants circulating among the population thereby serving as a proper surveillance method. The variant of concern (VOC) Alpha was first identified in September 2020 in the United Kingdom, and rapidly became dominant across Europe. Our objective was to elucidate the Alpha VOC outcompetition rate and identify mutations in the spike glycoprotein (S) gene, indicative of the circulation of the Alpha VOC and/or other variants in the population through wastewater analysis. In the period covered by this study (November 2020-April 2021), forteen wastewater treatment plants (WWTPs) were weekly sampled. The total number of SARS-CoV-2 genome copies per L (GC/L) was determined with a Real-Time qPCR, targeting the N gene. Surveillance of the Alpha VOC circulation was ascertained using a duplex RT-qPCR, targeting and discriminating the S gene. Our results showed that in a period of 6 weeks the Alpha VOC was present in all the studied WWTPs, and became dominant in 11 weeks on average. The outcompetition rates of the Alpha VOC were estimated, and their relationship with different parameters statistically analyzed. The rapid spread of the Alpha VOC was influenced by its initial input and by the previous circulation of SARS-COV-2 in the population. This latter point could be explained by its higher transmissibility, particularly advantadgeous when a certain degree of herd immunity exists. Moreover, the presence of signature mutations of SARS-COV-2 variants were established by deep-sequencing of the complete S gene. The circulation of the Alpha VOC in the area under study was confirmed, and additionally two combinations of mutations in the S glycoprotein (T73A and D253N, and S477N and A522S) that could affect antibody binding were identified

Definition of a list of fish diseases to aid health management in Spain

Trabajo presentado en la 14th EAFP International Conference (European Association of Fish Pathologists), celebrada en Praga (República Checa), del 14 al 19 de septiembre de 2009An expert working group used a risk ranking technique to compile a list of fish disease hazards (pathogens) of relevance to Spanish aquaculture. It was possible to divide the list into three groups: I-high national risk; II-regional risk; III-low risk. The three groups were as follows: Group I (high risk) Aphanomyces invadans (EUS)**, spring viraemia of carp virus (SVCV), koi herpes virus (KHV)¿ and infectious haematopoietic necrosis virus (IHNV)¿ Group II (regional risk) Enteromyxum spp. (leei and scophthalmi), Aquabirnaviridae (incl. IPNV), viral encephalopathy and retinopathy virus (VERV), Streptococcus iniae, Philasterides dicentrarchi and Aeromonas salmonicida (in the marine environment). Group III (low risk) Sparicotyle chrysophrii/Microcotylidae, Flavobacterium maritimus, Photobacterium piscicida, Togaviridae, Sphaerospora testicularis, Edwardsiella tarda, Birnavirus (no-EVE), Lactococcus garviae, viral haemorrhagic septicaemia virus (VHSV)¿, Tenacibaculum maritimum, epizootic haematopoietic necrosis virus (EHNV)**, Renibacterium salmoninarum (BKD) and Gyrodactylus salaris. The exercise formed part of a project concerning aquaculture health management (Jacumar-GESAC¿) in Spain and is being used to help define sampling plans for disease monitoring using epidemiological and risk-based criteria. **The diseases caused by these pathogens are notifiable and exotic according to Directive 2006/88/EC ¿The diseases caused by these pathogens are notifiable and non-exotic according to Directive 2006/88/EC ¿Plan Nacional de Cultivos Marinos; Gestión sanitaria de la acuicultura: Adaptación a la nueva normativa (GESAC) ¿ financed by the Junta Nacional Asesora de Cultivos Marinos (Jacumar

The Catalan Surveillance Network of SARS-CoV-2 in Sewage: design, implementation, and performance

Wastewater-based epidemiology has shown to be an efficient tool to track the circulation of SARS-CoV-2 in communities assisted by wastewater treatment plants (WWTPs). The challenge comes when this approach is employed to help Health authorities in their decision-making. Here, we describe the roadmap for the design and deployment of SARSAIGUA, the Catalan Surveillance Network of SARS-CoV-2 in Sewage. The network monitors, weekly or biweekly, 56 WWTPs evenly distributed across the territory and serving 6 M inhabitants (80% of the Catalan population). Each week, samples from 45 WWTPs are collected, analyzed, results reported to Health authorities, and finally published within less than 72 h in an online dashboard ( https://sarsaigua.icra.cat ). After 20 months of monitoring (July 20-March 22), the standardized viral load (gene copies/day) in all the WWTPs monitored fairly matched the cumulative number of COVID-19 cases along the successive pandemic waves, showing a good fit with the diagnosed cases in the served municipalities (Spearman Rho = 0.69). Here we describe the roadmap of the design and deployment of SARSAIGUA while providing several open-access tools for the management and visualization of the surveillance data.The authors wish to thank the staff from all the WWTPs monitored for their help and technical support during the sampling campaigns. The authors acknowledge the funding received from the ACA and the ASPCAT from the Catalan Government (Generalitat de Catalunya). ICRA authors acknowledge the funding provided by the Generalitat de Catalunya through the Consolidated Research Group grants ICRA-ENV 2017 SGR 1124 and ICRA-TiA 2017 SGR 1318. ICRA researchers also thank the funding from the CERCA program of the Catalan Government.Peer reviewe

High archaeal richness in the water column of a freshwater sulfurous karstic lake along an interannual study.

12 páginas, 2 tablas, 5 figures.We surveyed the archaeal assemblage in a stratified sulfurous lake (Lake Vilar, Banyoles, Spain) over 5 consecutive years to detect potential seasonal and interannual trends in the free-living planktonic Archaea composition. The combination of different primer pairs and nested PCR steps revealed an unexpectedly rich archaeal community. Overall, 140 samples were analyzed, yielding 169 different 16S rRNA gene sequences spread over 14 Crenarchaeota (109 sequences) and six Euryarchaeota phylogenetic clusters. Most of the Crenarchaeota (98% of the total crenarchaeotal sequences) affiliated within the Miscellaneous Crenarchaeota Group (MCG) and were related to both marine and freshwater phylotypes. Euryarchaeota mainly grouped within the Deep Hydrothermal Vent Euryarchaeota (DHVE) cluster (80% of the euryarchaeotal sequences) and the remaining 20% distributed into three less abundant taxa, most of them composed of soil and sediment clones. The largest fraction of phylotypes from the two archaeal kingdoms (79% of the Crenarchaeota and 54% of the Euryarchaeota) was retrieved from the anoxic hypolimnion, indicating that these cold and sulfide-rich waters constitute an unexplored source of archaeal richness. The taxon rank-frequency distribution showed two abundant taxa (MCG and DHVE) that persisted in the water column through seasons, plus several rare ones that were only detected occasionally. Differences in richness distribution and seasonality were observed, but no clear correlations were obtained when multivariate statistical analyses were carried out.This study was funded through projects VIARC (Ref. REN 2003-08333-GLO) and CRENYC (Ref. CGL2006-12058-BOS) to C.M.B. and E.O.C. from the Spanish Ministerio de Educación y Ciencia (MEC). M.L. is recipient of a PhD student fellowship (BES-2004-5127) from the Spanish MEC.Peer reviewe

The molar extinction coefficient of bacteriochlorophyll e and the pigment stoichiometry in Chlorobium phaeobacteroides

We have determined the molar extinction coefficient of bacteriochlorophyll (BChl) e, the main light-harvesting pigment from brown-coloured photosynthetic sulfur bacteria. The extinction coefficient was determined using pure[Pr,E]BChl eF isolated by reversed-phase HPLC from crude pigment extracts of Chlorobium (Chl.) phaeobacteroides strain CL1401. The extinction coefficients at the Soret and Qy bands were determined in four organic solvents. The extinction coefficient of BChl e differs from those of other related Chlorobium chlorophylls (BChl c and BChl d) but is similar to that of chlorophyll b. The determined extinction coefficient was used to calculate the stoichiometric BChl e to BChl a and BChl e to carotenoids ratios in whole cells and isolated chlorosomes from Chl. phaeobacteroides strain CL1401 using the spectrum-reconstruction method (SRCM) described by Naqvi et al. (1997) (Spectrochim Acta A Mol Biomol Spectrosc 53: 2229–2234). In isolated chlorosomes, BChl a content was ca. 1% of the total BChl content and the stoichiometric ratio of BChl e to carotenoids was 6. In whole cells,however, BChl a content was 3–4%, owing to the presence of BChl a-containing elements, i.e. FMO protein and reaction centre. An average of 5 BChl e molecules per carotenoid was determined in whole cells.EU(Contract No FMRX–CT96–0081). Ministerio de Educación y Ciencia (Ref. BIO96–1229–002–01)Peer reviewe