II Jornada de Joves Investigadors de l’IdRA

La I Jornada de Joves investigadors és una primera iniciativa per fer aflorar i donar visibilitat a la recerca dels més joves, planter de les futures generacions de l'IdRA. La pròpia tasca organitzativa i científica de na Julia Martín Díaz i na Laura Sala Comorera constitueix un bon exponent de la qualitat científica i del dinamisme dels investigadors més joves de l'IdRA. La Dra. Roser Rubio ha aportat, així mateix, la seva experiència i, amb la Julia i la Laura han estat l'exemple de la perfecta integració de dues generacions diferents unides pel seu compromís amb la ciència

Implementation and integration of microbial source tracking in a river watershed monitoring plan

Fecal pollution of water bodies poses a serious threat for public health and ecosystems. Microbial source tracking (MST) is used to track the source of this pollution facilitating better management of pollution at the source. In this study we tested 12 MST markers to track human, ruminant, sheep, horse, pig and gull pollution to assess their usefulness as an effective management tool of water quality. First, the potential of the selected markers to track the source was evaluated using fresh fecal samples. Subsequently, we evaluated their performance in a catchment with different impacts, considering land use and environmental conditions. All MST markers showed high sensitivity and specificity, although none achieved 100% for both. Although some of the MST markers were detected in hosts other than the intended ones, their abundance in the target group was always several orders of magnitude higher than in the non-target hosts, demonstrating their suitability to distinguish between sources of pollution. The MST analysis matched the land use in the watershed allowing an accurate assessment of the main sources of pollution, in this case mainly human and ruminant pollution. Correlating environmental parameters including temperature and rainfall with MST markers provided insight into the dynamics of the pollution in the catchment. The levels of the human marker showed a significant negative correlation with rainfall in human polluted areas suggesting a dilution of the pollution, whereas at agricultural areas the ruminant marker increased with rainfall. There were no seasonal differences in the levels of human marker, indicating human pollution as a constant pressure throughout the year, whereas the levels of the ruminant marker was influenced by the seasons, being more abundant in summer and autumn. MST analysis integrated with land use and environmental data can improve the management of fecal polluted areas and set up best practice

Traceability of different brands of bottled mineral water during shelf life, using PCR-DGGE and next generation sequencing techniques

Natural mineral waters contain indigenous bacteria characteristic of each spring source. Once bottled, these communities change over time until the water is consumed. Bottle material is believed to play a major role in the succession of these populations, but very few studies to date have evaluated the effect of this material on bacterial communities. In this study, we examined the microbial community structure of three natural mineral waters over 3 months after bottling in glass and polyethylene terephthalate (PET) bottles. To this end, we used culture-dependent (heterotrophic plate count) and culture-independent methods (16S rRNA massive gene sequencing, denaturing gradient gel electrophoresis (DGGE) and fluorescent microscopy with vital dyes). Total and viable cell counts increased by around 1-2 log10 units between 1 and 2 weeks after bottling and then remained constant over 3 months for all waters regardless of the bottle material. DGGE fingerprints and 16S rRNA massive sequencing analysis both indicated that different communities were established in the waters two weeks after bottling in the different bottle materials. In conclusion, no differences in total, viable and culturable bacteria counts were observed between mineral waters bottled with PET or glass during shelf life storage. Nevertheless, in spite of changes in the communities, each water brand and material presented a distinct microbial community structure clearly distinguishable from the others, which could be interesting for traceability purposes

Unravelling the composition of tap and mineral water microbiota: Divergences between next-generation sequencing techniques and culture-based methods

The complex and highly diverse microbial environment of drinking water, consisting mainly of bacteria at different metabolic states, is still underexplored. The aim of this work was to characterize the bacterial communities in tap water and bottled mineral water, the two predominant sources of drinking water in modern societies. A total of 11 tap water samples from a range of locations and distribution networks and 10 brands of bottled natural mineral water were analysed using two approaches: a) heterotrophic plate counts by matrixassisted laser desorption/ionization time of flight mass-spectrometry (MALDI-TOF MS) for the culturable heterotrophic communities, and b) Illumina amplicon sequencing for total bacteria including non-culturable bacteria. Culturable heterotrophic bacteria were isolated in WPCA (ISO) agar at 22 ± 2 °C for 72 h and 2046 isolates were identified using MALDI-TOF MS. The Bruker Daltonics Library and a previously customized library (Drinking Water Library) were used as reference databases. For the total bacteria fraction, DNA was extracted from 6 L of water and submitted to Illumina 16S rRNA sequencing of the v4 region. Significant differences were observed between mineral and tap water, with a general dominance of Alphaproteobacteria (mainly the genus Blastomonas) in tap water and Gammaproteobacteria in mineral water with Acidovorax being the dominant genus in 3 out of 7 mineral water brands. The bacterial communities in the different brands of mineral water were highly diverse and characteristic of each one. Moreover, the season in which the water was bottled also affected the species distribution, with some of them identified in only one season. Among the culturable bacteria, the most abundant phylum was Proteobacteria (around 85% of the isolates), followed by Actinobacteria, Firmicutes and Bacteroidetes. Proteobacteria was also the most abundant phylum detected with Illumina sequencing (> 99% of the reads). The two methods gave distinct results at the different taxonomic levels and could therefore have a complimentary application in the study of microbiota in mineral water environments. MALDI-TOF MS is a promising method for the rapid identification of heterotrophic bacteria in routine water analysis in the bottling industry. Significance and impact of the study: The complementarity of MALDI-TOF MS and NGS in the assessment of bacterial community diversity has been demonstrated in water intended for human consumption. The two methods are suitable for routine use in the water industry for water quality management

Rapid and improved identification of drinking water bacteria using the Drinking Water Library, a dedicated MALDI-TOF MS database

According to the European Directives (UE) 2020/2184 and 2009/54/EC, which establishes the sanitary criteria for water intended for human consumption in Europe, water suitable for human consumption must be free of the bacterial indicators Escherichia coli, Clostridium perfringens and Enterococcus spp. Drinking water is also monitored for heterotrophic bacteria, which are not a human health risk, but can serve as an index of bacteriological water quality. Therefore, a rapid, accurate, and cost-effective method for the identification of these colonies would improve our understanding of the culturable bacteria of drinking water and facilitate the task of water management by treatment facilities. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is potentially such a method, although most of the currently available mass spectral libraries have been developed in a clinical setting and have limited environmental applicability. In this work, a MALDITOF MS drinking water library (DWL) was defined and developed by targeting bacteria present in water intended for human consumption. This database, made up of 319 different bacterial strains, can contribute to the routine microbiological control of either treated drinking water or mineral bottled water carried out by water treatment and distribution operators, offering a faster identification rate compared to a clinical sample-based library. The DWL, made up of 96 bacterial genera, 44 of which are not represented in the MALDI-TOF MS bacterial Bruker Daltonics (BDAL) database, was found to significantly improve the identification of bacteria present in drinking water

Drinking Water Library: nuevo recurso para identificar cepas bacterianas en aguas de consumo

De acuerdo con el RD 140/2003, el agua apta para el consumo humano no debe mostrar presencia de los indicadores bacterianos Escherichia coli, Clostridium perfringens y Enterococcus sp. Pero, aunque no se detecten estos indicadores, el agua del grifo no es estéril y puede tener cierta cantidad de microrganismos que muchas veces puede generar colonias en las placas de cultivo. Lejos de representar un peligro para la salud humana, si se dispone de un método rápido, sencillo, robusto y fiable para la identificación de estas colonias, se puede avanzar mucho en el conocimiento de la microbiología del agua potable. El proyecto Drinking Water Library ha permitido desarrollar la primera base de datos para MALDI-TOF MS específica para bacterias presentes en aguas de consumo

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

Wastewater surveillance for pathogens using the reverse transcription-polymerase chain reaction (RT-PCR) is an effective, resource-efficient tool for gathering additional community-level public health information, including the incidence and/or prevalence and trends of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater may provide an early-warning signal of COVID-19 infections in a community. The capacity of the world’s environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is rapidly increasing. However, there are no standardized protocols nor harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can lead to false-positive and -negative errors in the surveillance of SARS-CoV-2, culminating in recommendations and strategies that can be implemented to identify and mitigate these errors. Recommendations include, stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly during a low incidence of SARS-CoV-2 in wastewater. Corrective and confirmatory actions must be in place for inconclusive and/or potentially significant results (e.g., initial onset or reemergence of COVID-19 in a community). It will also be prudent to perform inter-laboratory comparisons to ensure results are reliable and interpretable for ongoing and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance was demonstrated during this global crisis. In the future, wastewater will play an important role in the surveillance of a range of other communicable diseases.Highlights: Harmonized QA/QC procedures for SARS-CoV-2 wastewater surveillance are lacking; Wastewater analysis protocols are not optimized for trace analysis of viruses; False-positive and -negative errors have consequences for public health responses; Inter-laboratory studies utilizing standardized reference materials and protocols are needed.info:eu-repo/semantics/publishedVersio

Diversitat de comunitats heteròtrofes associades a les aigües de consum

[cat] Els indicadors microbians de la contaminació fecal han estat útils al llarg dels anys pel control sanitari de la qualitat de l’aigua de consum. Tot i així, l’aigua mineral embotellada i l’aigua de la xarxa de distribució no són aigües estèrils sinó que contenen una gran diversitat de microorganismes. De fet, la diversitat pròpia de l’aigua de consum ha estat poc explorada fins el moment. El propòsit d’aquesta tesi doctoral va ser contribuir a l’estudi de la composició i la dinàmica de les comunitats microbianes de diferents tipus d’aigua de consum humà: aigua mineral natural envasada i aigua potable de distribució al llarg del procés de potabilització. Referent a les aigües minerals naturals envasades, es va estudiar el canvi en les comunitats heteròtrofes de dues aigües minerals envasades en ampolles de vidre durant la seva vida comercial, fins a un any després de l’envasat. La comunitat microbiana heteròtrofa en placa no va ser estable durant la comercialització de l'aigua, sinó que presentava fluctuacions. Es va observar una disminució de la diversitat microbiana i dels recomptes d’heteròtrofs després d'un any. D’altra banda es va avaluar la traçabilitat de l’aigua mineral envasada amb tècniques dependents i no dependents de cultiu durant els 3 mesos després de l’envasat. Per això, es va realitzar un seguiment en 3 aigües minerals diferents envasades al mateix dia en PET i vidre. El nombre total de cèl·lules i de cèl·lules viables va augmentar durant les dues primeres setmanes després de l’envasat. A partir del dia 21, no es van observar diferències significatives en els recomptes entre les ampolles de diferents materials. En canvi, les empremtes moleculars per a cada aigua i material d’ampolla en alguns casos van ser característiques durant els 3 mesos. En l’àmbit de l’aigua de xarxa de distribució es va valorar la utilització de la tecnologia emergent MALDI-TOF MS per a l'ús en rutina per a la identificació de soques aïllades en una ETAP (estacions de tractament d’aigua potable). Aquesta tècnica es va comparar amb el sistema PhenePlateTM i amb les galeries API®. El percentatge de soques identificades amb el MALDI-TOF MS a nivell d'espècie (39%) va ser molt similar al percentatge obtingut amb les galeries API® (43%). A més, la utilització de MALDI-TOF MS va ser comparable amb el sistema PhenePlateTM (80%). Així doncs, la identificació mitjançant MALDI-TOF MS és una metodologia útil i prometedora en els laboratoris de rutina d'anàlisi d'aigua, gràcies a la seva robustesa i rapidesa per a la identificació microbiana. No obstant això, els espectres proteics de soques ambientals existents a les bases de dades són limitats. A partir de la identificació de soques aïllades en controls rutinaris en una ETAP mitjançant MALDI-TOF MS permet obtenir una caracterització ràpida de les comunitats heteròtrofes presents al llarg dels tractaments de l'aigua. Finalment aquesta tesi es va centrar en l’estudi de les comunitats planctòniques de l’aigua abans i després del tractament d’osmosi inversa (OI) d’una ETAP. L'estudi va rebel·lar que existien diferències entre la composició bacteriana entre els dos punts, indicant una correcta filtració a nivell d'OI i una posterior recolonització de l'aigua osmotitzada. En cap cas, es va detectar el patogen oportunista Pseudomonas aeruginosa. D'altra banda, la presència de soques amb capacitat per formar biofilm com també la presència de soques resistents a antibiòtics pot contribuir al biofouling i a la dispersió de resistències a antibiòtics. En conseqüència, es fa palès que les aigües minerals naturals envasades són microsistemes complexos. D’altra banda, la informació de les comunitats microbianes obtingudes en anàlisis rutinaris permetria millorar la gestió de les plantes.[eng] Natural mineral water and drinking water are not sterile environments, but complex ecosystems with a high phenotypic and genetic diversity of autochthonous bacteria. The purpose of this thesis was to contribute to the study of the composition and dynamics of the microbial communities in natural mineral water and drinking water. Regarding natural mineral water, it was studied the variations in the heterotrophic communities in two mineral waters bottled in glass bottles during their shelf life up to one year after bottling. The heterotrophic plate counts were not constant during the commercialization of the water, but they fluctuated. There was a decrease in the diversity of the heterotrophic plate counts after one year. Moreover, it was assessed the traceability of bottled mineral water with culture-dependent and culture-independent techniques during three months after packaging in three different mineral waters bottled in glass and PET. The number of total and viable cell counts increased during the first two weeks after the bottling and the molecular fingerprints for some mineral water brands and bottle material were different during the three months. As far as drinking water is concerned, we assessed the use of the emerging technology MALDI-TOF MS for routine identification of isolates in a drinking water treatment plant (DWTP). This technique was compared with the PhenePlateTM system and API® galleries. The percentage of strains identified with MALDI-TOF MS at species level (39%) was similar to the percentage obtained with API® galleries (43%). In addition, the use of MALDI-TOF MS system was reliable compared to PhenePlateTM (80%). Thus, the identification by MALDI-TOF MS is a promising methodology useful in routine laboratories thanks to its robustness and speed for microbial identification. The major limiting factor for MALDI–TOF MS is the lack of a suitable mass spectra database. Finally, this thesis focused on the study of the planktonic communities of feed and permeate water in a reverse osmosis treatment in a DWTP. The results revealed that there were differences in the bacterial composition between them, a successful level filtration via reverse osmosis and subsequent recolonization of the membrane with different species from those in the feed water was found

I Jornada de Joves Investigadors de l'IdRA

La I Jornada de Joves investigadors és una primera iniciativa per fer aflorar i donar visibilitat a la recerca dels més joves, planter de les futures generacions de l'IdRA. La pròpia tasca organitzativa i científica de na Julia Martín Díaz i na Laura Sala Comorera constitueix un bon exponent de la qualitat científica i del dinamisme dels investigadors més joves de l'IdRA. La Dra. Roser Rubio ha aportat, així mateix, la seva experiència i, amb la Julia i la Laura han estat l'exemple de la perfecta integració de dues generacions diferents unides pel seu compromís amb la ciència