Detection of quorum-sensing-related molecules in Vibrio scophthalmi

BACKGROUND: Cell-to-cell communication (also referred to as quorum sensing) based on N-acyl-homoserine lactones (AHLs) is a widespread response to environmental change in Gram-negative bacteria. AHLs seem to be highly variable, both in terms of the acyl chain length and in the chemical structure of the radicals. Another quorum sensing pathway, the autoinducer-2-based system, is present both in Gram-positive and Gram-negative bacteria. In this study the presence of signal molecules belonging to both quorum sensing signalling pathways was analysed in the marine symbiotic species Vibrio scophthalmi. RESULTS: Three AHL-like signal molecules were detected in V. scophthalmi supernatants with the Agrobacterium tumefaciens sensor assay. This observation was further supported by the decrease in the presence of these signal molecules after cloning and expression of lactonase AiiA from Bacillus cereus in the V. scophthalmi strains. One of the signal molecules was identified as N-(3-hydroxy dodecanoyl)-L-homoserine lactone. V. scophthalmi was also shown to carry a functional LuxS synthase. The coding sequence for a luxS-like gene was obtained showing a maximum similarity of 78% with Vibrio vulnificus. Analysis of the translated sequence revealed that the sequenced luxS gene carried the conserved domain, which is common to luxS sequences found in other species, and which is essential for LuxS enzymatic activity. CONCLUSION: The data are consistent with the presence of quorum-sensing signal molecules from both AHL- and autoinducer 2-based quorum sensing systems in V. scophthalmi, which are homologous to others previously described in various Vibrio species. How this bacterium interacts with other bacteria and eukaryotic cells to compete ecologically with other intestinal bacteria present in the fish Scophthalmus maximus warrants further investigation

Quorum-sensing regulates biofilm formation in Vibrio scophthalmi

Background: In a previous study, we demonstrated that Vibrio scophthalmi, the most abundant Vibrio species among the marine aerobic or facultatively anaerobic bacteria inhabiting the intestinal tract of healthy cultured turbot (Scophthalmus maximus), contains at least two quorum-sensing circuits involving two types of signal molecules (a 3-hydroxy-dodecanoyl-homoserine lactone and the universal autoinducer 2 encoded by luxS). The purpose of this study was to investigate the functions regulated by these quorum sensing circuits in this vibrio by constructing mutants for the genes involved in these circuits. Results. The presence of a homologue to the Vibrio harveyi luxR gene encoding a main transcriptional regulator, whose expression is modulated by quorumsensing signal molecules in other vibrios, was detected and sequenced. The V. scophthalmi LuxR protein displayed a maximum amino acid identity of 82% with SmcR, the LuxR homologue found in Vibrio vulnificus. luxR and luxS null mutants were constructed and their phenotype analysed. Both mutants displayed reduced biofilm formation in vitro as well as differences in membrane protein expression by mass-spectrometry analysis. Additionally, a recombinant strain of V. scophthalmi carrying the lactonase AiiA from Bacillus cereus, which causes hydrolysis of acyl homoserine lactones, was included in the study. Conclusions: V. scophthalmi shares two quorum sensing circuits, including the main transcriptional regulator luxR, with some pathogenic vibrios such as V. harveyi and V. anguillarum. However, contrary to these pathogenic vibrios no virulence factors (such as protease production) were found to be quorum sensing regulated in this bacterium. Noteworthy, biofilm formation was altered in luxS and luxR mutants. In these mutants a different expression profile of membrane proteins were observed with respect to the wild type strain suggesting that quorum sensing could play a role in the regulation of the adhesion mechanisms of this bacterium

Culture and molecular methods as complementary tools for water quality management

Bacterial communities in a full-scale drinking water treatment plant (DWTP) were characterized using matrix-assisted laser desorption/ionization time of flight mass-spectrometry (MALDI-TOF MS) to identify HPC isolates and the obtained results were compared to 16S rRNA (V4) metabarcoding data acquired in a previous study. Sixty-three samples were collected at nine stages of the potabilization process: river water and groundwater intake, decantation, sand filtration, ozonization, carbon filtration, reverse osmosis, the mixing chamber and post-chlorination drinking water. In total, 1807 bacterial colonies were isolated, 32 % of which were successfully identified to at least the genus level by MALDI-TOF MS using our previously developed Drinking Water Library. Trends in diversity were similar by both approaches, but differences were observed in the detection of taxa, especially at lower hierarchy levels. High bacterial diversity was observed in river and groundwater, where Proteobacteria predominated. The diversity decreased significantly after the chlorination step, where Bacillus sp. (Firmicutes) and an unknown genus of Obscuribacteraceae (Cyanobacteria) were the most prevalent genera according to MALDI-TOF MS and metabarcoding, respectively. The two approaches gave similar results for the decantation, sand filtration and mixing chamber steps, where the most abundant taxon was Flavobacterium. The combined use of these culture-based and culture-independent methods to characterize microbial populations may help to better understand the role of bacteria in water treatment and quality, which will be of value for DWTP management

Determination of crAssphage in water samples and applicability for tracking human fecal pollution

In recent decades, considerable effort has been devoted to finding microbial source-tracking (MST) markers that are suitable to assess the health risks of faecally polluted waters, with no universal marker reported so far. In this study, the abundance and prevalence of a crAssphage-derived DNA marker in wastewaters of human and animal origins were studied by a new qPCR assay with the ultimate aim of assessing its potential as an MST marker. crAssphage showed up to 106 GC/ml in the sewage samples of human origin, in both the total DNA and the viral DNA fraction. In wastewaters containing animal faecal remains, 39% of the samples were negative for the presence of the crAssphage sequence, while those showing positive results (41% of the samples) were at least 1 log10 unit lower than the samples of human origin. Noteworthy, the log10 values of the ratio (R) crAssphage (GC/ml)/Escherichia coli (CFU/ml) varied significantly depending on the human or animal origin (R > 1.5 for human samples and R < −1.5 for animal wastewater samples. This study opens the way for further research to explore if different specific animal variants of crAssphage exist and whether other zones of the crAssphage genome are better suited to source discrimination

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

Monitoring bacterial community dynamics in a drinking water treatment plant: an integrative approach using metabarcoding and microbial indicators in large water volumes

Monitoring bacterial communities in a drinking water treatment plant (DWTP) may help to understand their regular operations. Bacterial community dynamics in an advanced full-scale DWTP were analyzed by 16S rRNA metabarcoding, and microbial water quality indicators were determined at nine different stages of potabilization: river water and groundwater intake, decantation, sand filtration, ozonization, carbon filtration, reverse osmosis, mixing chamber and post-chlorination drinking water. The microbial content of large water volumes (up to 1100 L) was concentrated by hollow fiber ultrafiltration. Around 10 million reads were obtained and grouped into 10,039 amplicon sequence variants. Metabarcoding analysis showed high bacterial diversity at all treatment stages and above all in groundwater intake, followed by carbon filtration and mixing chamber samples. Shifts in bacterial communities occurred downstream of ozonization, carbon filtration, and, more drastically, chlorination. Proteobacteria and Bacteroidota predominated in river water and throughout the process, but in the final drinking water, the strong selective pressure of chlorination reduced diversity and was clearly dominated by Cyanobacteria. Significant seasonal variation in species distribution was observed in decantation and carbon filtration samples. Some amplicon sequence variants related to potentially pathogenic genera were found in the DWTP. However, they were either not detected in the final water or in very low abundance (<2%), and all EU Directive quality standards were fully met. A combination of culture and high-throughput sequencing techniques may help DWTP managers to detect shifts in microbiome, allowing for a more in-depth assessment of operational performance

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

Impact of treated sewage effluent on the bacterial community composition in an intermittent mediterranean stream

Water quality monitoring is essential to safeguard human and environmental health. The advent of next-generation sequencing techniques in recent years, which allow a more in-depth study of environmental microbial communities in the environment, could broaden the perspective of water quality monitoring to include impact of faecal pollution bacteria on ecosystem. In this study, 16 S rRNA amplicon sequencing was used to evaluate the impact of wastewater treatment plant (WWTP) effluent on autochthonous microbial communities of a temporary Mediterranean stream characterized by high flow seasonality (from 0.02 m(3)/s in winter to 0.006 m(3)/s in summer). Seven sampling campaigns were performed under different temperatures and streamflow conditions (winter and summer). Water samples were collected upstream (Upper) of the WWTP, the secondary effluent (EF) discharge and 75 m (P75) and 1000 m (P1000) downstream of the WWTP. A total of 5,593,724 sequences were obtained, giving rise to 20,650 amplicon sequence variants (ASV), which were further analysed and classified into phylum, class, family and genus. Each sample presented different distribution and abundance of taxa. Although taxon distribution and abundance differed in each sample, the microbial community structure of P75 resembled that of EF samples, and Upper and P1000 samples mostly clustered together. Alpha diversity showed the highest values for Upper and P1000 samples and presented seasonal differences, being higher in winter conditions of high streamflow and low temperature. Our results suggest the microbial ecology re-establishment, since autochthonous bacterial communities were able to recover from the impact of the WWTP effluent in 1 km. Alpha diversity results indicates a possible influence of environmental factors on the bacterial community structure. This study shows the potential of next-generation sequencing techniques as useful tools in water quality monitoring and management within the climate change scenari

Experimental evidence of antimicrobial activity in Antarctic seaweeds: ecological role and antibiotic potential

Seaweeds contain a wide range of secondary metabolites which serve multiple functions, including chemical and ecological mediation with microorganisms. Moreover, owing to their diverse bioactivity, including their antibiotic properties, they show potential for human use. Nonetheless, the chemical ecology of seaweeds is not equally understood across different regions; for example, Antarctic seaweeds are among the lesser studied groups. With the aim of improving our current understanding of the chemical ecology and potential bioactivity of Antarctic seaweeds, we performed a screening of antibiotic activity using crude extracts from 22 Antarctic macroalgae species. Extractions were performed separating lipophilic and hydrophilic fractions at natural concentrations. Antimicrobial activity assays were performed using the disk diffusion method against seven Antarctic bacteria and seven human pathogenic surrogates. Our results showed that red seaweeds (especially Delisea pulchra) inhibited a larger number of microorganisms compared with brown seaweeds, and that lipophilic fractions were more active than hydrophilic ones. Both types of bacteria tested (Gram negative and Gram positive) were inhibited, especially by butanolic fractions, suggesting a trend of non-specific chemical defence. However, Gram-negative bacteria and one pathogenic fungus showed greater resistance. Our study contributes to the evidence of antimicrobial chemical interactions between Antarctic seaweeds and sympatric microorganisms, as well as the potential of seaweed extracts for pharmacological applications

Assessment of dead-end ultrafiltration for the detection and quantification of microbial indicators and pathogens in the drinking water treatment processes

A safe water supply requires distinct treatments and monitoring to guarantee the absence of pathogens and substances potentially hazardous for human health. In this study we assessed the efficiency of the dead-end ultrafiltration (DEUF) method to concentrate faecal indicator organisms (FIO) and pathogens in water samples with different physicochemical characteristics. Water samples were collected at the treatment stages of two drinking water treatment plants to analyse the concentration of a variety of 7 FIO and 4 reference microbes which have some species that are pathogenic to humans: Campylobacter spp., enteroviruses, Cryptosporidium spp. and Giardia spp. The samples were analysed before and after concentration by DEUF, detecting FIO concentrations about 1 log10 higher in non-concentrated samples from both catchments. Percent recoveries were highly variable with a mean of 43.8 ± 17.5%, depending on the FIO and inherent sample characteristics. However, DEUF enabled FIO concentration in high volumes of water (100-500 l), allowing a reduction in the detection limit compared to the non-concentrated samples due to the high volume processing capabilities of the method. As a consequence, the detection of FIO removal from water in the drinking water treatment process was 1.0-1.5 logarithms greater in DEUF-treated water compared to unfiltered samples. The DEUF method improved the detection of target indicators and allowed for the detection of pathogens in low concentrations in water after the treatment stages, confirming the suitability of DEUF to concentrate high volumes of different types of water. This method could be useful for microbial analysis in water treatment monitoring and risk assessment, allowing the identification of critical points during the water treatment process and potential hazards in water destined for several uses