From shells to sequences: A proof-of-concept study for on-site analysis of hemolymphatic circulating cell-free DNA from sentinel mussels using Nanopore technology.

Blue mussels are often abundant and widely distributed in polar marine coastal ecosystems. Because of their wide distribution, ecological importance, and relatively stationary lifestyle, bivalves have long been considered suitable indicators of ecosystem health and changes. Monitoring the population dynamics of blue mussels can provide information on the overall biodiversity, species interactions, and ecosystem functioning. In the present work, we combined the concept of liquid biopsy (LB), an emerging concept in medicine based on the sequencing of free circulating DNA, with the Oxford Nanopore Technologies (ONT) platform using a portable laboratory in a remote area. Our results demonstrate that this platform is ideally suited for sequencing hemolymphatic circulating cell-free DNA (ccfDNA) fragments found in blue mussels. The percentage of non-self ccfDNA accounted for >50 % of ccfDNA at certain sampling Sites, allowing the quick, on-site acquisition of a global view of the biodiversity of a coastal marine ecosystem. These ccfDNA fragments originated from viruses, bacteria, plants, arthropods, algae, and multiple Chordata. Aside from non-self ccfDNA, we found DNA fragments from all 14 blue mussel chromosomes, as well as those originating from the mitochondrial genomes. However, the distribution of nuclear and mitochondrial DNA was significantly different between Sites. Similarly, analyses between various sampling Sites showed that the biodiversity varied significantly within microhabitats. Our work shows that the ONT platform is well-suited for LB in sentinel blue mussels in remote and challenging conditions, enabling faster fieldwork for conservation strategies and resource management in diverse settings.

Divergent bacterial landscapes: unraveling geographically driven microbiomes in Atlantic cod.

Establishing microbiome signatures is now recognized as a critical step toward identifying genetic and environmental factors shaping animal-associated microbiomes and informing the health status of a given host. In the present work, we prospectively collected 63 blood samples of the Atlantic cod population of the Southern Gulf of Saint Lawrence (GSL) and characterized their 16S rRNA circulating microbiome signature. Our results revealed that the blood microbiome signature was dominated at the phylum level by Proteobacteria, Bacteroidetes, Acidobacteria and Actinobacteria, a typical signature for fish populations inhabiting the GSL and other marine ecosystems. At the genus level, however, we identified two distinct cod groups. While the microbiome signature of the first group was dominated by Pseudoalteromonas, a genus we previously found in the microbiome signature of Greenland and Atlantic halibut populations of the GSL, the second group had a microbiome signature dominated by Nitrobacter and Sediminibacterium (approximately 75% of the circulating microbiome). Cods harboring a Nitrobacter/Sediminibacterium-rich microbiome signature were localized in the most southern part of the GSL, just along the northern coast of Cape Breton Island. Atlantic cod microbiome signatures did not correlate with the weight, length, relative condition, depth, temperature, sex, and salinity, as previously observed in the halibut populations. Our study provides, for the first time, a unique snapshot of the circulating microbiome signature of Atlantic cod populations and the potential existence of dysbiotic signatures associated with the geographical distribution of the population, probably linked with the presence of nitrite in the environment.

ß-D-glucuronidase activity triggered monitoring of fecal contamination using microbial and chemical source tracking markers at drinking water intakes.

Intense rainfall and snowmelt events may affect the safety of drinking water, as large quantities of fecal material can be discharged from storm or sewage overflows or washed from the catchment into drinking water sources. This study used ß-d-glucuronidase activity (GLUC) with microbial source tracking (MST) markers: human, bovine, porcine mitochondrial DNA markers (mtDNA) and human-associated Bacteroidales HF183 and chemical source tracking (CST) markers including caffeine, carbamazepine, theophylline and acetaminophen, pathogens (Giardia, Cryptosporidium, adenovirus, rotavirus and enterovirus), water quality indicators (Escherichia coli, turbidity) and hydrometeorological data (flowrate, precipitation) to assess the vulnerability of 3 drinking water intakes (DWIs) and identify sources of fecal contamination. Water samples were collected under baseline, snow and rain events conditions in urban and agricultural catchments (Québec, Canada). Dynamics of E. coli, HF183 and WWMPs were similar during contamination events, and concentrations generally varied over 1 order of magnitude during each event. Elevated human-associated marker levels during events demonstrated that urban DWIs were impacted by recent contamination from an upstream municipal water resource recovery facility (WRRF). In the agricultural catchment, mixed fecal pollution was observed with the occurrences and increases of enteric viruses, human bovine and porcine mtDNA during peak contaminating events. Bovine mtDNA qPCR concentrations were indicative of runoff of cattle-derived fecal pollutants to the DWI from diffuse sources following rain events. This study demonstrated that the suitability of a given MST or CST indicator depend on river and catchment characteristics. The sampling strategy using continuous online GLUC activity coupled with MST and CST markers analysis was a more reliable source indicator than turbidity to identify peak events at drinking water intakes.

Assessment of multiple fecal contamination sources in surface waters using environmental mitochondrial DNA metabarcoding.

Waterborne diseases are transmitted to humans through the fecal contamination of water, where homeothermic species are the main reservoir. Fecal indicator bacteria (FIB) are often used to determine the occurrence of fecal contamination. However, FIB cannot provide the source of fecal contamination. Furthermore, as fecal inputs and contamination could originate from multiple sources (e.g., human, livestock, wildlife), multiple source tracking markers are required to identify fecal sources. From a previous study, we developed a mitochondrial DNA (mtDNA) metabarcoding approach to assess the presence of multiple homeotherms in four surface waters. Here, we have broadened our approach by sampling 86 surface water samples from the L'Assomption River and Ville-Marie watersheds (Province of Quebec, Canada). Fecal coliform levels were higher than the expected sanitary recommendations for recreational water (> 200 CFU/100 mL) in 73 % samples. The occurrence of mtDNA from human, livestock, domestic animals, wild mammals and wild birds was found in 40-88 % of the samples. Multivariate analyses showed significant covariations between homeothermic taxa and fecal coliforms, enterococci, ß-D-glucuronidase, conductivity, the human-specific Bacteroidales Hf183 genetic marker, and the human population, in the watersheds of L'Assomption River (p = 0.001) and Ville-Marie (p = 0.015) (Province of Quebec, Canada). Through the application of Bayes Theorem, it was determined that fecal coliforms co-occurred with the detection of bovine, beaver, robin and chicken mtDNA in 100 % of cases in the L'Assomption River watershed, and human mtDNA co-occurred with fecal coliforms in 93 % and 76 % of cases in L'Assomption River watershed and Ville-Marie sub-catchment, respectively. This study suggests that fecal contamination could be the result of multiple species, among which some wild animals may contribute to fecal inputs in surface waters, resulting in potential risk to human health. This reinforces the necessity of using the mtDNA metabarcoding method to monitor multi-animal species.

Insights into the circulating microbiome of Atlantic and Greenland halibut populations: the role of species-specific and environmental factors.

Establishing long-term microbiome-based monitoring programs is critical for managing and conserving wild fish populations in response to climate change. In most cases, these studies have been conducted on gut and, to a lesser extent, skin (mucus) microbiomes. Here, we exploited the concept of liquid biopsy to study the circulating bacterial microbiome of two Northern halibut species of economic and ecological importance. Amplification and sequencing of the 16S rRNA gene were achieved using a single drop of blood fixed on FTA cards to identify the core blood microbiome of Atlantic and Greenland halibut populations inhabiting the Gulf of St. Lawrence, Canada. We provide evidence that the circulating microbiome DNA (cmDNA) is driven by genetic and environmental factors. More specifically, we found that the circulating microbiome signatures are species-specific and vary according to sex, size, temperature, condition factor, and geographical localization. Overall, our study provides a novel approach for detecting dysbiosis signatures and the risk of disease in wild fish populations for fisheries management, most notably in the context of climate change.

Species- and site-specific circulating bacterial DNA in Subantarctic sentinel mussels Aulacomya atra and Mytilus platensis.

Impacts of climate changes are particularly severe in polar regions where warmer temperatures and reductions in sea-ice covers threaten the ecological integrity of marine coastal ecosystems. Because of their wide distribution and their ecological importance, mussels are currently used as sentinel organisms in monitoring programs of coastal ecosystems around the world. In the present study, we exploited the concept of liquid biopsy combined to a logistically friendly sampling method to study the hemolymphatic bacterial microbiome in two mussel species (Aulacomya atra and Mytilus platensis) in Kerguelen Islands, a remote Subantarctic volcanic archipelago. We found that the circulating microbiome signatures of both species differ significantly even though their share the same mussel beds. We also found that the microbiome differs significantly between sampling sites, often correlating with the particularity of the ecosystem. Predictive models also revealed that both species have distinct functional microbiota, and that the circulating microbiome of Aulacomya atra was more sensitive to changes induced by acute thermal stress when compared to Mytilus platensis. Taken together, our study suggests that defining circulating microbiome is a useful tool to assess the health status of marine ecosystems and to better understand the interactions between the sentinel species and their habitat.

Influence of temperature and water quality on the persistence of human mitochondrial DNA, human Hf183 Bacteroidales, fecal coliforms and enterococci in surface water in human fecal source tracking context.

Mitochondrial DNA (mtDNA) is used as a genetic marker to track fecal contamination in surface water. Its potential to effectively discriminate between the nonpoint sources of fecal pollution (e.g. human, livestock) in water environments is relevant for water quality management. However, there is a lack of knowledge about the environmental persistence of mtDNA in relation to those of other microbial parameters, such as fecal indicator bacteria (FIB). In this study, mesocosms composed of water collected from four rivers and tap water were spiked with raw wastewater to mimic human fecal contamination. Mesocosms composed of raw wastewater were also studied. The mesocosms were incubated at 4 °C or at 22 °C for 189 days, from which the levels of human mtDNA (HumtDNA) and human Bacteroidales (Hf183) were measured by qPCR. The levels of FIB (fecal coliforms and enterococci) and heterotrophs were determined by culture methods along with the determination of physicochemical attributes. The decay rates of the genetic markers and FIB were determined with first-order decay rate models. The decay rates of HumtDNA (0.004-0.059 d-1), Hf183 (0.007-0.082 d-1), and the two FIBs (0.005-0.066 d-1) were similar at 4 °C, while the genetic markers both had higher decay rates (0.013-0.919 d-1) at 22 °C. Different HumtDNA decay rates were observed between the river mesocosms (0.043-0.919 d-1) and the wastewater and tap water mesocosms (0.004-0.095 d-1). Covariations of pH and conductivity among the HumtDNA, Hf183 and FIB decay rates were observed. HumtDNA and Hf183 had similar environmental persistence, whereas fecal coliforms and enterococci persisted longer at 22 °C. Finally, HumtDNA had the same trends of persistence in the four river mesocosms, suggesting a relative stability of this marker in different rivers. Our results suggest that HumtDNA could be more suitable for tracking the source of a recent fecal contamination in complement to FIB.

eDNA profiling of mammals, birds, and fish of surface waters by mitochondrial metagenomics: application for source tracking of fecal contamination in surface waters.

Knowing the composition of animals present in aquatic ecosystems can tell us about the anthropic pressures on these environments. One of these pressures is the occurrence of fecal contamination. However, this contamination can originate from more than one animal species in areas where urban and agricultural activities overlap. Mitochondrial DNA (mtDNA) has become the standard barcoding tool to identify the presence of animal species in environment. Amplicon-sequencing metagenomics is a powerful approach to derive the animal profile in an environment. However, PCR primers targeting mtDNA of a broad range of animals are highly degenerate or generate short DNA fragments that could cause ambiguous affiliation. Here we report the development of a new set of primers targeting the mitochondrial 16S ribosomal RNA genes of a broad range of terrestrial and aquatic animals, which include mammals, birds, and fishes. These primers successfully amplified mtDNA from environmental DNA (eDNA) extracted from surface waters. Sequencing the resulting amplicons revealed the presence of mammals and birds that may contribute in fecal contamination of surface water. In one of the river samples high in fecal indicator bacteria, human and bovine mtDNA accounted for 40.5% and 4.1% of the sequences, respectively, suggesting fecal contamination by these two animals. These findings indicate that our PCR primers coupled with amplicon-sequencing metagenomics contribute in profiling the animal diversity in the surface waters and its surrounding. This approach could be a valuable tool to identify simultaneously the potential contribution of various animals as sources of fecal contamination in surface waters.

Applying the concept of liquid biopsy to monitor the microbial biodiversity of marine coastal ecosystems.

Liquid biopsy (LB) is a concept that is rapidly gaining ground in the biomedical field. Its concept is largely based on the detection of circulating cell-free DNA (ccfDNA) fragments that are mostly released as small fragments following cell death in various tissues. A small percentage of these fragments are from foreign (nonself) tissues or organisms. In the present work, we applied this concept to mussels, a sentinel species known for its high filtration capacity of seawater. We exploited the capacity of mussels to be used as natural filters to capture environmental DNA fragments of different origins to provide information on the biodiversity of marine coastal ecosystems. Our results showed that hemolymph of mussels contains DNA fragments that varied considerably in size, ranging from 1 to 5 kb. Shotgun sequencing revealed that a significant amount of DNA fragments had a nonself microbial origin. Among these, we found DNA fragments derived from bacteria, archaea, and viruses, including viruses known to infect a variety of hosts that commonly populate coastal marine ecosystems. Taken together, our study shows that the concept of LB applied to mussels provides a rich and yet unexplored source of knowledge regarding the microbial biodiversity of a marine coastal ecosystem.

Co-culturing Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 allows sustainable denitrifying activities under marine conditions.

BACKGROUND: Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 are the principal bacteria involved in the denitrifying activities of a methanol-fed, fluidized-bed marine denitrification system. Strain NL23 possesses the complete denitrification pathway, but cannot grow under marine conditions in pure cultures. Strain JAM1 is a marine bacterium that lacks genes encoding a dissimilatory nitrite (NO2 -) reductase and therefore cannot reduce NO2 -. Here, we report the characterization of some of their physiological traits that could influence their co-habitation. We also perform co-cultures to assess the potential synergy between the two strains under marine and denitrifying conditions. METHODOLOGY: Anoxic planktonic pure cultures of both strains were grown with different concentrations of nitrate (NO3 -). Anoxic planktonic co-cultures could only be cultured on low NaCl concentrations for strain NL23 to grow. Biofilm co-cultures were achieved in a 500-mL bioreactor, and operated under denitrifying conditions with increasing concentrations of NaCl. NO3 - and NO2 - concentrations and the protein content were measured to derive the denitrification rates. The concentrations of both strains in co-cultures were determined by quantitative PCR (qPCR). Ectoine concentration was measured by mass spectrometry in the biofilm co-culture. The biofilm was visualized by fluorescence in situ hybridization. Reverse-transcription-qPCR and RNA-seq approaches were used to assess changes in the expression profiles of genes involved in the nitrogen pathways in the biofilm cultures. RESULTS: Planktonic pure cultures of strain JAM1 had a readiness to reduce NO3 - with no lag phase for growth in contrast to pure cultures of strain NL23, which had a 2-3 days lag phase before NO3 - starts to be consumed and growth to occur. Compared to strain NL23, strain JAM1 has a higher µmax for growth and higher specific NO3 - reduction rates. Denitrification rates were twice higher in the planktonic co-cultures than those measured in strain NL23 pure cultures. The biofilm co-cultures showed sustained denitrifying activities and surface colonization by both strains under marine conditions. Increase in ectoine concentrations was observed in the biofilm co-culture with the increase of NaCl concentrations. Changes in the relative transcript levels were observed in the biofilm culture with genes encoding NapA and NapGH in strain NL23. The type of medium had a great impact on the expression of genes involved in the N-assimilation pathways in both strains. CONCLUSIONS: These results illustrate the capacity of both strains to act together in performing sustainable denitrifying activities under marine conditions. Although strain JAM1 did not contribute in better specific denitrifying activities in the biofilm co-cultures, its presence helped strain NL23 to acclimate to medium with NaCl concentrations >1.0%.

Correction: Liquid biopsies for omics-based analysis in sentinel mussels.

[This corrects the article DOI: 10.1371/journal.pone.0223525.].

Liquid biopsies for omics-based analysis in sentinel mussels.

Liquid biopsy of plasma is a simple and non-invasive technology that holds great promise in biomedical research. It is based on the analysis of nucleic acid-based biomarkers with predictive potential. In the present work, we have combined this concept with the FTA technology for sentinel mussels. We found that hemocytes collected from liquid biopsies can be readily fixed on FTA cards and used for long-term transcriptome analysis. We also showed that liquid biopsy is easily adaptable for metagenomic analysis of bacterial profiles of mussels. We finally provide evidence that liquid biopsies contained circulating cell-free DNA (ccfDNA) which can be used as an easily accessible genomic reservoir. Sampling of FTA-fixed circulating nucleic acids is stable at room temperature and does not necessitate a cold-chain protection. It showed comparable performance to frozen samples and is ideally adapted for sampling in remote areas, most notably in polar regions threatened by anthropogenic activities. From an ethical point of view, this minimally-invasive and non-lethal approach further reduces incidental mortality associated with conventional tissue sampling. This liquid biopsy-based approach should thus facilitate biobanking activities and development of omics-based biomarkers in mussels to assess the quality of aquatic ecosystems.

Dynamics of a methanol-fed marine denitrifying biofilm: 2-impact of environmental changes on the microbial community.

BACKGROUND: The biofilm of a methanol-fed, marine denitrification system is composed of a multi-species microbial community, among which Hyphomicrobium nitrativorans and Methylophaga nitratireducenticrescens are the principal bacteria involved in the denitrifying activities. To assess its resilience to environmental changes, the biofilm was cultivated in artificial seawater (ASW) under anoxic conditions and exposed to a range of specific environmental conditions. We previously reported the impact of these changes on the denitrifying activities and the co-occurrence of H. nitrativorans strain NL23 and M. nitratireducenticrescens in the biofilm cultures. Here, we report the impact of these changes on the dynamics of the overall microbial community of the denitrifying biofilm. METHODS: The original biofilm (OB) taken from the denitrification system was cultivated in ASW under anoxic conditions with a range of NaCl concentrations, and with four combinations of nitrate/methanol concentrations and temperatures. The OB was also cultivated in the commercial Instant Ocean seawater (IO). The bacterial diversity of the biofilm cultures and the OB was determined by 16S ribosomal RNA gene sequences. Culture approach was used to isolate other denitrifying bacteria from the biofilm cultures. The metatranscriptomes of selected biofilm cultures were derived, along with the transcriptomes of planktonic pure cultures of H. nitrativorans strain NL23 and M. nitratireducenticrescens strain GP59. RESULTS: High proportions of M. nitratireducenticrescens occurred in the biofilm cultures. H. nitrativorans strain NL23 was found in high proportion in the OB, but was absent in the biofilm cultures cultivated in the ASW medium at 2.75% NaCl. It was found however in low proportions in the biofilm cultures cultivated in the ASW medium at 0-1% NaCl and in the IO biofilm cultures. Denitrifying bacterial isolates affiliated to Marinobacter spp. and Paracoccus spp. were isolated. Up regulation of the denitrification genes of strains GP59 and NL23 occurred in the biofilm cultures compared to the planktonic pure cultures. Denitrifying bacteria affiliated to the Stappia spp. were metabolically active in the biofilm cultures. CONCLUSIONS: These results illustrate the dynamics of the microbial community in the denitrifying biofilm cultures in adapting to different environmental conditions. The NaCl concentration is an important factor affecting the microbial community in the biofilm cultures. Up regulation of the denitrification genes of M. nitratireducenticrescens strain GP59 and H. nitrativorans strain NL23 in the biofilm cultures suggests different mechanisms of regulation of the denitrification pathway in the biofilm. Other denitrifying heterotrophic bacteria are present in low proportions, suggesting that the biofilm has the potential to adapt to heterotrophic, non-methylotrophic environments.

Dynamics of a methanol-fed marine denitrifying biofilm: 1-Impact of environmental changes on the denitrification and the co-occurrence of Methylophaga nitratireducenticrescens and Hyphomicrobium nitrativorans.

BACKGROUND: The biofilm of a methanol-fed denitrification system that treated a marine effluent is composed of multi-species microorganisms, among which Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 are the principal bacteria involved in the denitrifying activities. Here, we report the capacity of the denitrifying biofilm to sustain environmental changes, and the impact of these changes on the co-occurrence of H. nitrativorans and M. nitratireducenticrescens. METHODS: In a first set of assays, the original biofilm (OB) was cultivated in an artificial seawater (ASW) medium under anoxic conditions to colonize new carriers. The new formed biofilm was then subjected to short exposures (1-5 days) of a range of NaCl, methanol, nitrate (NO3 -) and nitrite (NO2 -) concentrations, and to different pHs and temperatures. In a second set of assays, the OB was cultivated in ASW medium for five weeks with (i) a range of NaCl concentrations, (ii) four combinations of NO3 -/methanol concentrations and temperatures, (iii) NO2 -, and (iv) under oxic conditions. Finally, the OB was cultivated for five weeks in the commercial Instant Ocean (IO) seawater. The growth of the biofilm and the dynamics of NO3 - and NO2 - were determined. The levels of M. nitratireducenticrescens and H. nitrativorans were measured by qPCR. RESULTS: In the first set of assays, the biofilm cultures had the capacity to sustain denitrifying activities in most of the tested conditions. Inhibition occurred when they were exposed to high pH (10) or to high methanol concentration (1.5%). In the second set of assays, the highest specific denitrification rates occurred with the biofilm cultures cultivated at 64.3 mM NO3 - and 0.45% methanol, and at 30 °C. Poor biofilm development occurred with the biofilm cultures cultivated at 5% and 8% NaCl. In all biofilm cultures cultivated in ASW at 2.75% NaCl, H. nitrativorans strain NL23 decreased by three orders of magnitude in concentrations compared to that found in OB. This decrease coincided with the increase of the same magnitude of a subpopulation of M. nitratireducenticrescens (strain GP59 as representative). In the biofilm cultures cultivated at low NaCl concentrations (0% to 1.0%), persistence of H. nitrativorans strain NL23 was observed, with the gradual increase in concentrations of M. nitratireducenticrescens strain GP59. High levels of H. nitrativorans strain NL23 were found in the IO biofilm cultures. The concentrations of M. nitratireducenticrescens strain JAM1 were lower in most of the biofilms cultures than in OB. CONCLUSIONS: These results demonstrate the plasticity of the marine methylotrophic denitrifying biofilm in adapting to different environmental changes. The NaCl concentration is a crucial factor in the dynamics of H. nitrativorans strain NL23, for which growth was impaired above 1% NaCl in the ASW-based biofilm cultures in favor of M. nitratireducenticrescens strain GP59.

Fecal contamination of storm sewers: Evaluating wastewater micropollutants, human-specific Bacteroides 16S rRNA, and mitochondrial DNA genetic markers as alternative indicators of sewer cross connections.

A set of fecal indicator bacteria and alternative markers were tested for their use to identify priority sectors of two urban watersheds in the Greater Montreal region with unintended household sewage connections to storm drainage systems. Analyses were performed for thermotolerant (fecal) coliforms (FC), Escherichia coli, human-specific Bacteroidales (HF183) and mitochondrial DNA (Hmt) markers, carbamazepine (CBZ), caffeine (CAF), theophylline (THEO) and acetaminophen (ACE). A high incidence of human fecal contamination was observed, illustrating the need for a method to appropriately prioritize sectors for the rehabilitation of sewer cross-connections. Concentrations of alternative markers were not significantly different between the residential and industrial/commercial/institutional (ICI) sectors. However, median E. coli concentrations were higher in the residential as compared to ICI sectors (p < 0.05). Hmt marker, CAF, and THEO were well correlated to E. coli in the ICI sector (r > 0.61, p < 0.05). Considering all sites, only CAF and THEO were correlated to E. coli (r > 0.59, p < 0.05), possibly as a result of higher E. coli inputs from other sources such as domestic animals or fauna in the residential sector. Thresholds were determined to relate alternative markers to E. coli for use in an index for prioritizing sectors with sewer cross-connections. HF183, Hmt, CAF, THEO, and ACE were identified as suitable markers for identifying sewer cross-connections and are more reliable than E. coli alone, most importantly in residential sectors.

Strain-level genetic diversity of Methylophaga nitratireducenticrescens confers plasticity to denitrification capacity in a methylotrophic marine denitrifying biofilm.

BACKGROUND: The biofilm of a methanol-fed, fluidized denitrification system treating a marine effluent is composed of multi-species microorganisms, among which Hyphomicrobium nitrativorans NL23 and Methylophaga nitratireducenticrescens JAM1 are the principal bacteria involved in the denitrifying activities. Strain NL23 can carry complete nitrate (NO[Formula: see text]) reduction to N2, whereas strain JAM1 can perform 3 out of the 4 reduction steps. A small proportion of other denitrifiers exists in the biofilm, suggesting the potential plasticity of the biofilm in adapting to environmental changes. Here, we report the acclimation of the denitrifying biofilm from continuous operating mode to batch operating mode, and the isolation and characterization from the acclimated biofilm of a new denitrifying bacterial strain, named GP59. METHODS: The denitrifying biofilm was batch-cultured under anoxic conditions. The acclimated biofilm was plated on Methylophaga specific medium to isolate denitrifying Methylophaga isolates. Planktonic cultures of strains GP59 and JAM1 were performed, and the growth and the dynamics of NO[Formula: see text], nitrite (NO[Formula: see text]) and N2O were determined. The genomes of strains GP59 and JAM1 were sequenced and compared. The transcriptomes of strains GP59 and JAM1 were derived from anoxic cultures. RESULTS: During batch cultures of the biofilm, we observed the disappearance of H. nitrativorans NL23 without affecting the denitrification performance. From the acclimated biofilm, we isolated strain GP59 that can perform, like H. nitrativorans NL23, the complete denitrification pathway. The GP59 cell concentration in the acclimated biofilm was 2-3 orders of magnitude higher than M. nitratireducenticrescens JAM1 and H. nitrativorans NL23. Genome analyses revealed that strain GP59 belongs to the species M. nitratireducenticrescens. The GP59 genome shares more than 85% of its coding sequences with those of strain JAM1. Based on transcriptomic analyses of anoxic cultures, most of these common genes in strain GP59 were expressed at similar level than their counterparts in strain JAM1. In contrast to strain JAM1, strain GP59 cannot reduce NO[Formula: see text] under oxic culture conditions, and has a 24-h lag time before growth and NO[Formula: see text] reduction start to occur in anoxic cultures, suggesting that both strains regulate differently the expression of their denitrification genes. Strain GP59 has the ability to reduce NO[Formula: see text] as it carries a gene encoding a NirK-type NO[Formula: see text] reductase. Based on the CRISPR sequences, strain GP59 did not emerge from strain JAM1 during the biofilm batch cultures but rather was present in the original biofilm and was enriched during this process. DISCUSSION: These results reinforce the unique trait of the species M. nitratireducenticrescens among the Methylophaga genus as facultative anaerobic bacterium. These findings also showed the plasticity of denitrifying population of the biofilm in adapting to anoxic marine environments of the bioreactor.

Denitrifying metabolism of the methylotrophic marine bacterium Methylophaga nitratireducenticrescens strain JAM1.

BACKGROUND: Methylophaga nitratireducenticrescens strain JAM1 is a methylotrophic, marine bacterium that was isolated from a denitrification reactor treating a closed-circuit seawater aquarium. It can sustain growth under anoxic conditions by reducing nitrate ([Formula: see text]) to nitrite ([Formula: see text]). These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (Nar). Strain JAM1 also contains gene clusters encoding two nitric oxide (NO) reductases and one nitrous oxide (N2O) reductase, suggesting that NO and N2O can be reduced by strain JAM1. Here we characterized further the denitrifying activities of M. nitratireducenticrescens JAM1. METHODS: Series of oxic and anoxic cultures of strain JAM1 were performed with N2O, [Formula: see text] or sodium nitroprusside, and growth and N2O, [Formula: see text], [Formula: see text] and N2 concentrations were measured. Ammonium ([Formula: see text])-free cultures were also tested to assess the dynamics of N2O, [Formula: see text] and [Formula: see text]. Isotopic labeling of N2O was performed in 15NH4+-amended cultures. Cultures with the JAM1ΔnarG1narG2 double mutant were performed to assess the involvement of the Nar systems on N2O production. Finally, RT-qPCR was used to measure the gene expression levels of the denitrification genes cytochrome bc-type nitric oxide reductase (cnorB1 and cnorB2) and nitrous oxide reductase (nosZ), and also nnrS and norR that encode NO-sensitive regulators. RESULTS: Strain JAM1 can reduce NO to N2O and N2O to N2 and can sustain growth under anoxic conditions by reducing N2O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory [Formula: see text] reductase, [Formula: see text]-amended cultures produce N2O, representing up to 6% of the N-input. [Formula: see text] was shown to be the key intermediate of this production process. Upregulation in the expression of cnorB1, cnorB2, nnrS and norR during the growth and the N2O accumulation phases suggests NO production in strain JAM1 cultures. DISCUSSION: By showing that all the three denitrification reductases are active, this demonstrates that M. nitratireducenticrescens JAM1 is one of many bacteria species that maintain genes associated primarily with denitrification, but not necessarily related to the maintenance of the entire pathway. The reason to maintain such an incomplete pathway could be related to the specific role of strain JAM1 in the denitrifying biofilm of the denitrification reactor from which it originates. The production of N2O in strain JAM1 did not involve Nar, contrary to what was demonstrated in Escherichia coli. M. nitratireducenticrescens JAM1 is the only reported Methylophaga species that has the capacity to grow under anoxic conditions by using [Formula: see text] and N2O as sole electron acceptors for its growth. It is also one of a few marine methylotrophs that is studied at the physiological and genetic levels in relation to its capacity to perform denitrifying activities.

Detection of virulence, antibiotic resistance and toxin (VAT) genes in Campylobacter species using newly developed multiplex PCR assays.

Campylobacter species are one of the leading causes of bacterial gastroenteritis in humans worldwide. This twofold study was sought to: i) develop and optimize four single-tube multiplex PCR (mPCR) assays for the detection of six virulence (ciaB, dnaJ, flaA, flaB, pldA and racR), three toxin (cdtA, cdtB and cdtC) and one antibiotic resistance tet(O) genes in thermophilic Campylobacter spp. and ii) apply and evaluate the developed mPCR assays by testing 470 previously identified C. jejuni, C. coli and C. lari isolates from agricultural water. In each mPCR assay, a combination of two or three sets of primer pairs for virulence, antibiotic resistance and toxin (VAT) genes was used and optimized. Assay 1 was developed for the detection of dnaJ, racR and cdtC genes with expected amplification sizes of 720, 584 and 182bp. Assay 2 generated PCR amplicons for tet(O) and cdtA genes of 559 and 370bp. Assay 3 amplified cdtB ciaB, and pldA genes with PCR amplicon sizes of 620, 527 and 385bp. Assay 4 was optimized for flaA and flaB genes that generated PCR amplicons of 855 and 260bp. The primer pairs and optimized PCR protocols did not show interference and/or cross-amplification with each other and generated the expected size of amplification products for each target VAT gene for the C. jejuni ATCC 33291 reference strain. Overall, all ten target VAT genes were detected at a variable frequency in tested isolates of thermophilic Campylobacter spp. where cdtC, flaB, ciaB, cdtB, cdtA and pldA were commonly detected compared to the flaA, racR, dnaJ and tet(O) genes which were detected with less frequency. The developed mPCR assays are simple, rapid, reliable and sensitive tools for simultaneously assessing potential pathogenicity and antibiotic resistance profiling in thermophilic Campylobacter spp. The mPCR assays will be useful in diagnostic and analytical settings for routine screening of VAT characteristics of Campylobacter spp. as well as being applicable in epidemiological studies by providing information that could be related to the risk of human infection.

Identification, characterization and description of Arcobacter faecis sp. nov., isolated from a human waste septic tank.

A study on the taxonomic classification of Arcobacter species was performed on the cultures isolated from various fecal sources where an Arcobacter strain AF1078(T) from human waste septic tank near Ottawa, Ontario, Canada was characterized using a polyphasic approach. Genetic investigations including 16S rRNA, atpA, cpn60, gyrA, gyrB and rpoB gene sequences of strain AF1078(T) are unique in comparison with other arcobacters. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain is most closely related to Arcobacter lanthieri and Arcobacter cibarius. Analyses of atpA, cpn60, gyrA, gyrB and rpoB gene sequences suggested that strain AF1078(T) formed a phylogenetic lineage independent of other species in the genus. Whole-genome sequence, DNA-DNA hybridization, fatty acid profile and phenotypic analysis further supported the conclusion that strain AF1078(T) represents a novel Arcobacter species, for which the name Arcobacter faecis sp. nov. is proposed, with type strain AF1078(T) (=LMG 28519(T); CCUG 66484(T)).

Development and evaluation of multiplex PCR assays for rapid detection of virulence-associated genes in Arcobacter species.

As the pathogenicity of Arcobacter species might be associated with various virulence factors, this study was aimed to develop and optimize three single-tube multiplex PCR (mPCR) assays that can efficiently detect multiple virulence-associated genes (VAGs) in Arcobacter spp. including the Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, respectively. The recognized target virulence factors used in the study were fibronectin binding protein (cj1349), filamentous hemagglutinin (hecA), hemolysin activation protein (hecB), hemolysin (tlyA), integral membrane protein virulence factor (mviN), invasin (ciaB), outer membrane protein (irgA) and phospholipase (pldA). Identical results were obtained between singleplex PCR and mPCR assays and no cross- and/or non-specific amplification products were obtained when tested against other closely related bacterial species. The sensitivities of these three mPCR assays were ranging from 1ngµL(-1) to 100ngµL(-1) DNA. The developed assays with combinations of duplex or triplex PCR primer pairs of VAGs were further evaluated and validated by applying them to isolates of the A. butzleri, A. cryaerophilus and A. skirrowii recovered from fecal samples of human and animal origins. The findings revealed that the distribution of the ciaB (90%), mviN (70%), tlyA (50%) and pldA (45%) genes among these target species was significantly higher than the hecA (16%), hecB (10%) and each of irgA and cj1349 (6%) genes, respectively. The newly developed mPCR assays can be used as rapid technique and useful markers for the detection, prevalence and profiling of VAGs in the Arcobacter spp. Moreover, these assays can easily be performed with a high throughput to give a presumptive identification of the causal pathogen in epidemiological investigation of human infections.