Plasmid permissiveness of wastewater microbiomes can be predicted from 16S rRNA sequences by machine learning.

MOTIVATION: Wastewater treatment plants (WWTPs) harbor a dense and diverse microbial community. They constantly receive antimicrobial residues and resistant strains, and therefore provide conditions for horizontal gene transfer (HGT) of antimicrobial resistance (AMR) determinants. This facilitates the transmission of clinically important genes between, e.g. enteric and environmental bacteria, and vice versa. Despite the clinical importance, tools for predicting HGT remain underdeveloped. RESULTS: In this study, we examined to which extent water cycle microbial community composition, as inferred by partial 16S rRNA gene sequences, can predict plasmid permissiveness, i.e. the ability of cells to receive a plasmid through conjugation, based on data from standardized filter mating assays using fluorescent bio-reporter plasmids. We leveraged a range of machine learning models for predicting the permissiveness for each taxon in the community, representing the range of hosts a plasmid is able to transfer to, for three broad host-range resistance IncP plasmids (pKJK5, pB10, and RP4). Our results indicate that the predicted permissiveness from the best performing model (random forest) showed a moderate-to-strong average correlation of 0.49 for pB10 [95% confidence interval (CI): 0.44-0.55], 0.43 for pKJK5 (0.95% CI: 0.41-0.49), and 0.53 for RP4 (0.95% CI: 0.48-0.57) with the experimental permissiveness in the unseen test dataset. Predictive phylogenetic signals occurred despite the broad host-range nature of these plasmids. Our results provide a framework that contributes to the assessment of the risk of AMR pollution in wastewater systems. AVAILABILITY AND IMPLEMENTATION: The predictive tool is available as an application at https://github.com/DaneshMoradigaravand/PlasmidPerm.

Characterization and Abundance of Plasmid-Dependent Alphatectivirus Bacteriophages.

Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis.

CRISPR-Cas systems are widespread accessory elements across bacterial and archaeal plasmids.

Many prokaryotes encode CRISPR-Cas systems as immune protection against mobile genetic elements (MGEs), yet a number of MGEs also harbor CRISPR-Cas components. With a few exceptions, CRISPR-Cas loci encoded on MGEs are uncharted and a comprehensive analysis of their distribution, prevalence, diversity, and function is lacking. Here, we systematically investigated CRISPR-Cas loci across the largest curated collection of natural bacterial and archaeal plasmids. CRISPR-Cas loci are widely but heterogeneously distributed across plasmids and, in comparison to host chromosomes, their mean prevalence per Mbp is higher and their distribution is distinct. Furthermore, the spacer content of plasmid CRISPRs exhibits a strong targeting bias towards other plasmids, while chromosomal arrays are enriched with virus-targeting spacers. These contrasting targeting preferences highlight the genetic independence of plasmids and suggest a major role for mediating plasmid-plasmid conflicts. Altogether, CRISPR-Cas are frequent accessory components of many plasmids, which is an overlooked phenomenon that possibly facilitates their dissemination across microbiomes.

Horizontal transmission of a multidrug-resistant IncN plasmid isolated from urban wastewater.

Wastewater treatment plants (WWTPs) are considered reservoirs of antibiotic resistance genes (ARGs). Given that plasmid-mediated horizontal gene transfer plays a critical role in disseminating ARGs in the environment, it is important to inspect the transfer potential of transmissible plasmids to have a better understanding of whether these mobile ARGs can be hosted by opportunistic pathogens and should be included in One Health's considerations. In this study, we used a fluorescent-reporter-gene based exogenous isolation approach to capture extended-spectrum beta-lactamases encoding mobile determinants from sewer microbiome samples that enter an urban water system (UWS) in Denmark. After screening and sequencing, we isolated a ∼73 Kbp IncN plasmid (pDK_DARWIN) that harboured and expressed multiple ARGs. Using a dual fluorescent reporter gene system, we showed that this plasmid can transfer into resident urban water communities. We demonstrated the transfer of pDK_DARWIN to microbiome members of both the sewer (in the upstream UWS compartment) and wastewater treatment (in the downstream UWS compartment) microbiomes. Sequence similarity search across curated plasmid repositories revealed that pDK_DARWIN derives from an IncN backbone harboured by environmental and nosocomial Enterobacterial isolates. Furthermore, we searched for pDK_DARWIN sequence matches in UWS metagenomes from three countries, revealing that this plasmid can be detected in all of them, with a higher relative abundance in hospital sewers compared to residential sewers. Overall, this study demonstrates that this IncN plasmid is prevalent across Europe and an efficient vector capable of disseminating multiple ARGs in the urban water systems.

Hordeum vulgare differentiates its response to beneficial bacteria.

BACKGROUND: In nature, beneficial bacteria triggering induced systemic resistance (ISR) may protect plants from potential diseases, reducing yield losses caused by diverse pathogens. However, little is known about how the host plant initially responds to different beneficial bacteria. To reveal the impact of different bacteria on barley (Hordeum vulgare), bacterial colonization patterns, gene expression, and composition of seed endophytes were explored. RESULTS: This study used the soil-borne Ensifer meliloti, as well as Pantoea sp. and Pseudomonas sp. isolated from barley seeds, individually. The results demonstrated that those bacteria persisted in the rhizosphere but with different colonization patterns. Although root-leaf translocation was not observed, all three bacteria induced systemic resistance (ISR) against foliar fungal pathogens. Transcriptome analysis revealed that ion- and stress-related genes were regulated in plants that first encountered bacteria. Iron homeostasis and heat stress responses were involved in the response to E. meliloti and Pantoea sp., even if the iron content was not altered. Heat shock protein-encoding genes responded to inoculation with Pantoea sp. and Pseudomonas sp. Furthermore, bacterial inoculation affected the composition of seed endophytes. Investigation of the following generation indicated that the enhanced resistance was not heritable. CONCLUSIONS: Here, using barley as a model, we highlighted different responses to three different beneficial bacteria as well as the influence of soil-borne Ensifer meliloti on the seed microbiome. In total, these results can help to understand the interaction between ISR-triggering bacteria and a crop plant, which is essential for the application of biological agents in sustainable agriculture.

Biosolids for safe land application: does wastewater treatment plant size matters when considering antibiotics, pollutants, microbiome, mobile genetic elements and associated resistance genes?

Soil fertilization with wastewater treatment plant (WWTP) biosolids is associated with the introduction of resistance genes (RGs), mobile genetic elements (MGEs) and potentially selective pollutants (antibiotics, heavy metals, disinfectants) into soil. Not much data are available on the parallel analysis of biosolid pollutant contents, RG/MGE abundances and microbial community composition. In the present study, DNA extracted from biosolids taken at 12 WWTPs (two large-scale, six middle-scale and four small-scale plants) was used to determine the abundance of RGs and MGEs via quantitative real-time PCR and the bacterial and archaeal community composition was assessed by 16S rRNA gene amplicon sequencing. Concentrations of heavy metals, antibiotics, the biocides triclosan, triclocarban and quaternary ammonium compounds (QACs) were measured. Strong and significant correlations were revealed between several target genes and concentrations of Cu, Zn, triclosan, several antibiotics and QACs. Interestingly, the size of the sewage treatment plant (inhabitant equivalents) was negatively correlated with antibiotic concentrations, RGs and MGEs abundances and had little influence on the load of metals and QACs or the microbial community composition. Biosolids from WWTPs with anaerobic treatment and hospitals in their catchment area were associated with a higher abundance of potential opportunistic pathogens and higher concentrations of QACs.

IncHI1A plasmids potentially facilitate horizontal flow of antibiotic resistance genes to pathogens in microbial communities of urban residential sewage.

Horizontal gene transfer via plasmids is important for the dissemination of antibiotic resistance genes among medically relevant pathogens. Specifically, the transfer of IncHI1A plasmids is believed to facilitate the spread of antibiotic resistance genes, such as carbapenemases, within the clinically important family Enterobacteriaceae. The microbial community of urban wastewater treatment plants has been shown to be highly permissive towards conjugal transfer of IncP1 plasmids. Here, we tracked the transfer of the P1 plasmid pB10 and the clinically relevant HI1A plasmid R27 in the microbial communities present in urban residential sewage entering full-scale wastewater treatment plants. We found that both plasmids readily transferred to these communities and that strains in the sewage were able to further disseminate them. Furthermore, R27 has a broad potential host range, but a low host divergence. Interestingly, although the majority of R27 transfer events were to members of Enterobacteriaceae, we found a subset of transfer events to other families, even other phyla. This indicates that HI1A plasmids facilitate horizontal gene transfer both within Enterobacteriaceae, but also across families of, in particular, Gammaproteobacteria, such as Moraxellaceae, Pseudomonadaceae and Shewanellaceae. pB10 displayed a similar potential host range to R27. In contrast to R27, pB10 had a high host divergence. By culture enrichment of the transconjugant communities, we show that sewage strains of Enterobacteriaceae and Aeromonadaceae can stably maintain R27 and pB10, respectively. Our results suggest that dissemination in the urban residual water system of HI1A plasmids may result in an accelerated acquisition of antibiotic resistance genes among pathogens.

Extended-Spectrum ß-Lactamase and Carbapenemase Genes are Substantially and Sequentially Reduced during Conveyance and Treatment of Urban Sewage.

Urban wastewater systems (UWSs) are a main receptacle of excreted antibiotic resistance genes (ARGs) and their host microorganisms. However, we lack integrated and quantitative observations of the occurrence of ARGs in the UWS to characterize the sources and identify processes that contribute to their fate. We sampled the UWSs from three medium-size cities in Denmark, Spain, and the United Kingdom and quantified 70 clinically important extended-spectrum ß-lactamase and carbapenemase genes along with the mobile genetic elements and microbial communities. Results from all three countries showed that sewage-especially from hospitals-carried substantial loads of ARGs (106-107 copies per person equivalent), but these loads progressively declined along sewers and through sewage treatment plants, resulting in minimal emissions (101-104 copies per person equivalent). Removal was primarily during sewage conveyance (65 ± 36%) rather than within sewage treatment (34 ± 23%). The extended-spectrum ß-lactamase and carbapenemase genes were clustered in groups based on their persistence in the UWS compartments. The less-persistent groups were associated to putative host taxa (especially Enterobacteriaceae and Moraxellaceae), while the more persistent groups appeared horizontally transferred and correlated significantly with total cell numbers and mobile genetic elements. This documentation of a substantial ARG reduction during sewage conveyance provides opportunities for antibiotic resistance management and a caution for sewage-based antibiotic resistance surveillance.

Salmonella persistence in soil depends on reciprocal interactions with indigenous microorganisms.

Fresh fruits and vegetables have numerous benefits to human health. Unfortunately, their consumption is increasingly associated with food-borne diseases, Salmonella enterica being their most frequent cause in Europe. Agricultural soils were postulated as reservoir of human pathogens, contributing to the contamination of crops during the growing period. Since the competition with the indigenous soil microbiota for colonization sites plays a major role in the success of invading species, we hypothesized that reduced diversity will enhance the chance of Salmonella to successfully establish in agricultural environments. We demonstrated that the abundance of Salmonella drastically decreased in soil with highly diverse indigenous prokaryotic community, while in soil with reduced prokaryotic diversity, Salmonella persisted for a long period. Furthermore, in communities with low diversity, Salmonella had an impact on the abundance of other taxa. The high physiological plasticity allows Salmonella to use agricultural soils as alternative habitat which might provide a route of animal/human infections. In addition, adjusted transcriptional profile with amino acid biosynthesis and the glyoxylate cycle most prominently regulated, suggests an adaptation to the soil environment. Our results underline the importance of the maintenance of diverse soil microbiome as a part of strategy aiming at reduced risk of food-borne salmonellosis outbreaks.

Fate of CMY-2-Encoding Plasmids Introduced into the Human Fecal Microbiota by Exogenous Escherichia coli.

The gut is a hot spot for transfer of antibiotic resistance genes from ingested exogenous bacteria to the indigenous microbiota. The objective of this study was to determine the fate of two nearly identical blaCMY-2-harboring plasmids introduced into the human fecal microbiota by two Escherichia coli strains isolated from a human and from poultry meat. The chromosome and the CMY-2-encoding plasmid of both strains were labeled with distinct fluorescent markers (mCherry and green fluorescent protein [GFP]), allowing fluorescence-activated cell sorting (FACS)-based tracking of the strain and the resident bacteria that have acquired its plasmid. Each strain was introduced into an established in vitro gut model (CoMiniGut) inoculated with individual feces from ten healthy volunteers. Fecal samples collected 2, 6, and 24 h after strain inoculation were analyzed by FACS and plate counts. Although the human strain survived better than the poultry meat strain, both strains transferred their plasmids to the fecal microbiota at concentrations as low as 102 CFU/ml. Strain survival and plasmid transfer varied significantly depending on inoculum concentration and individual fecal microbiota. Identification of transconjugants by 16S rRNA gene sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) revealed that the plasmids were predominantly acquired by Enterobacteriaceae species, such as E. coli and Hafnia alvei Our experimental data demonstrate that exogenous E. coli of human or animal origin can readily transfer CMY-2-encoding IncI1 plasmids to the human fecal microbiota. Small amounts of the exogenous strain are sufficient to ensure plasmid transfer if the strain is able to survive the gastric environment.

Characterization of Extracellular Biosurfactants Expressed by a Pseudomonas putida Strain Isolated from the Interior of Healthy Roots from Sida hermaphrodita Grown in a Heavy Metal Contaminated Soil.

Pseudomonas putida E41 isolated from root interior of Sida hermaphrodita (grown on a field contaminated with heavy metals) showed high biosurfactant activity. In this paper, we describe data from mass spectrometry and genome analysis, to improve our understanding on the phenotypic properties of the strain. Supernatant derived from P. putida E41 liquid culture exhibited a strong decrease in the surface tension accompanied by the ability for emulsion stabilization. We identified extracellular lipopeptides, putisolvin I and II expression but did not detect rhamnolipids. Their presence was confirmed by matrix-assisted laser desorption and ionization (MALDI) TOF/TOF technique. Moreover, ten phospholipids (mainly phosphatidylethanolamines PE 33:1 and PE 32:1) which were excreted by vesicles were also detected. In contrast the bacterial cell pellet was dominated by phosphatidylglycerols (PGs), which were almost absent in the supernatant. It seems that the composition of extracellular (secreted to the environment) and cellular lipids in this strain differs. Long-read sequencing and complete genome reconstruction allowed the identification of a complete putisolvin biosynthesis pathway. In the genome of P. putida E41 were also found all genes involved in glycerophospholipid biosynthesis, and they are likely responsible for the production of detected phospholipids. Overall this is the first report describing the expression of extracellular lipopeptides (identified as putisolvins) and phospholipids by a P. putida strain, which might be explained by the need to adapt to the highly contaminated environment.

The soil resistome: a critical review on antibiotic resistance origins, ecology and dissemination potential in telluric bacteria.

Soil is a large reservoir of microbial diversity and the majority of antimicrobial compounds used today in human and veterinary health care have been isolated from soil microorganisms. The Darwinian hypothesis of an 'arms-shields race' between antibiotic producers and resistant strains is often cited to explain antibiotic resistance gene determinants (ARGD) origins and diversity. ARGD abundance and antibiotic molecule exposure are, however, not systematically linked, and many other factors can contribute to resistance gene emergence, selection and dissemination in the environment. Soil is a heterogeneous habitat and represents a broad spectrum of different ecological niches. Soil harbours a large genetic diversity at small spatial scale, favouring exchange of genetic materials by means of horizontal gene transfer (HGT) that will contribute to ARGD dissemination between bacteria and eventually acquisition by pathogen genomes, therefore threatening antibiotic therapies. Our current knowledge on the extent of the soil resistome abundance and diversity has been greatly enhanced since the metagenomic revolution and help of high-throughput sequencing technologies. Different ecological hypotheses explaining their high prevalence in soil and questioning their transfer rate to pathogens, in respect to these recent experimental results, will be discussed in the present review.

A comparison of short-read, HiFi long-read, and hybrid strategies for genome-resolved metagenomics.

Shotgun metagenomics enables the reconstruction of complex microbial communities at a high level of detail. Such an approach can be conducted using both short-read and long-read sequencing data, as well as a combination of both. To assess the pros and cons of these different approaches, we used 22 fecal DNA extracts collected weekly for 11 weeks from two respective lab mice to study seven performance metrics over four combinations of sequencing depth and technology: (i) 20 Gbp of Illumina short-read data, (ii) 40 Gbp of short-read data, (iii) 20 Gbp of PacBio HiFi long-read data, and (iv) 40 Gbp of hybrid (20 Gbp of short-read +20 Gbp of long-read) data. No strategy was best for all metrics; instead, each one excelled across different metrics. The long-read approach yielded the best assembly statistics, with the highest N50 and lowest number of contigs. The 40 Gbp short-read approach yielded the highest number of refined bins. Finally, the hybrid approach yielded the longest assemblies and the highest mapping rate to the bacterial genomes. Our results suggest that while long-read sequencing significantly improves the quality of reconstructed bacterial genomes, it is more expensive and requires deeper sequencing than short-read approaches to recover a comparable amount of reconstructed genomes. The most optimal strategy is study-specific and depends on how researchers assess the trade-off between the quantity and quality of recovered genomes.IMPORTANCEMice are an important model organism for understanding the gut microbiome. When studying these gut microbiomes using DNA techniques, researchers can choose from technologies that use short or long DNA reads. In this study, we perform an extensive benchmark between short- and long-read DNA sequencing for studying mice gut microbiomes. We find that no one approach was best for all metrics and provide information that can help guide researchers in planning their experiments.

Interspecific interactions facilitate keystone species in a multispecies biofilm that promotes plant growth.

Microorganisms colonizing plant roots co-exist in complex, spatially structured multispecies biofilm communities. However, little is known about microbial interactions and the underlying spatial organization within biofilm communities established on plant roots. Here, a well-established four-species biofilm model (Stenotrophomonas rhizophila, Paenibacillus amylolyticus, Microbacterium oxydans, and Xanthomonas retroflexus, termed as SPMX) was applied to Arabidopsis roots to study the impact of multispecies biofilm on plant growth and the community spatial dynamics on the roots. SPMX co-culture notably promoted root development and plant biomass. Co-cultured SPMX increased root colonization and formed multispecies biofilms, structurally different from those formed by monocultures. By combining 16S rRNA gene amplicon sequencing and fluorescence in situ hybridization with confocal laser scanning microscopy, we found that the composition and spatial organization of the four-species biofilm significantly changed over time. Monoculture P. amylolyticus colonized plant roots poorly, but its population and root colonization were highly enhanced when residing in the four-species biofilm. Exclusion of P. amylolyticus from the community reduced overall biofilm production and root colonization of the three species, resulting in the loss of the plant growth-promoting effects. Combined with spatial analysis, this led to identification of P. amylolyticus as a keystone species. Our findings highlight that weak root colonizers may benefit from mutualistic interactions in complex communities and hereby become important keystone species impacting community spatial organization and function. This work expands the knowledge on spatial organization uncovering interspecific interactions in multispecies biofilm communities on plant roots, beneficial for harnessing microbial mutualism promoting plant growth.

Insights into the circular: The cryptic plasmidome and its derived antibiotic resistome in the urban water systems.

Plasmids have been a concern in the dissemination and evolution of antibiotic resistance in the environment. In this study, we investigated the total pool of plasmids (plasmidome) and its derived antibiotic resistance genes (ARGs) in different compartments of urban water systems (UWSs) in three European countries representing different antibiotic usage regimes. We applied a direct plasmidome approach using wet-lab methods to enrich circular DNA in the samples, followed by shotgun sequencing and in silico contig circularisation. We identified 9538 novel sequences in a total of 10,942 recovered circular plasmids. Of these, 66 were identified as conjugative, 1896 mobilisable and 8970 non-mobilisable plasmids. The UWSs' plasmidome was dominated by small plasmids (≤10 Kbp) representing a broad diversity of mobility (MOB) types and incompatibility (Inc) groups. A shared collection of plasmids from different countries was detected in all treatment compartments, and plasmids could be source-tracked in the UWSs. More than half of the ARGs-encoding plasmids carried mobility genes for mobilisation/conjugation. The richness and abundance of ARGs-encoding plasmids generally decreased with the flow, while we observed that non-mobilisable ARGs-harbouring plasmids maintained their abundance in the Spanish wastewater treatment plant. Overall, our work unravels that the UWS plasmidome is dominated by cryptic (i.e., non-mobilisable, non-typeable and previously unknown) plasmids. Considering that some of these plasmids carried ARGs, were prevalent across three countries and could persist throughout the UWSs compartments, these results should alarm and call for attention.

Co-localization of antibiotic resistance genes is widespread in the infant gut microbiome and associates with an immature gut microbial composition.

BACKGROUND: In environmental bacteria, the selective advantage of antibiotic resistance genes (ARGs) can be increased through co-localization with genes such as other ARGs, biocide resistance genes, metal resistance genes, and virulence genes (VGs). The gut microbiome of infants has been shown to contain numerous ARGs, however, co-localization related to ARGs is unknown during early life despite frequent exposures to biocides and metals from an early age. RESULTS: We conducted a comprehensive analysis of genetic co-localization of resistance genes in a cohort of 662 Danish children and examined the association between such co-localization and environmental factors as well as gut microbial maturation. Our study showed that co-localization of ARGs with other resistance and virulence genes is common in the early gut microbiome and is associated with gut bacteria that are indicative of low maturity. Statistical models showed that co-localization occurred mainly in the phylum Proteobacteria independent of high ARG content and contig length. We evaluated the stochasticity of co-localization occurrence using enrichment scores. The most common forms of co-localization involved tetracycline and fluoroquinolone resistance genes, and, on plasmids, co-localization predominantly occurred in the form of class 1 integrons. Antibiotic use caused a short-term increase in mobile ARGs, while non-mobile ARGs showed no significant change. Finally, we found that a high abundance of VGs was associated with low gut microbial maturity and that VGs showed even higher potential for mobility than ARGs. CONCLUSIONS: We found that the phenomenon of co-localization between ARGs and other resistance and VGs was prevalent in the gut at the beginning of life. It reveals the diversity that sustains antibiotic resistance and therefore indirectly emphasizes the need to apply caution in the use of antimicrobial agents in clinical practice, animal husbandry, and daily life to mitigate the escalation of resistance. Video Abstract.

Draft genomes of seven isolates from Danish wastewater facilities belonging to Pseudomonas, Bacillus, Pseudochrobactrum, Brevundimonas, and Pandoraea.

We report here seven draft genomes of bacterial strains from two Danish wastewater facilities, two of which might be characterized as a new group within the Pseudomonas and Pseudochrobactrum genera, respectively. These genomes will provide useful references for understanding bacterial interactions and horizontal gene transfer within bacterial communities.

Quantification and fate of plasmid-specific bacteriophages in wastewater: Beyond the F-coliphages.

Plasmid-specific bacteriophages specifically infect bacteria carrying conjugal plasmids. While wastewater has been used as isolation source for such phages, to date, only the distribution and ecology of RNA phages specific to the F plasmid have been described, because they serve as a water quality indicator. Yet, several other plasmid classes have higher clinical and ecological relevance, and the distribution, fate, and ecology of the phages that target them remain uncharacterized. We aimed to (i) provide an experimental platform to quantify the abundance of plasmid-specific phages applicable to several different conjugal plasmid classes, (ii) describe the distribution of such phages in wastewater systems, and (iii) relate their abundance to plasmid abundance and to municipal wastewater treatment processes. We introduced four model conjugal plasmids, belonging to incompatibility groups IncP-1, IncN, IncHI1, or IncF into an avirulent Salmonella enterica strain, for which somatic phages are at low abundance in wastewater. These strains were used in double layer agar assays with waters from contrasting sources. Plasmid-specific phages were common in wastewater but rare in river water. Hospital wastewater contained significantly more IncP-1-, but fewer IncF- and IncN- specific phages than domestic wastewater. This pattern did not match that of plasmid abundance estimated by Inc group targeting high-throughput quantitative PCR. The comparison between influent and effluent of wastewater treatment plants revealed a reduction in phage concentration by ca. 2 log, without significant contribution of primary settling. Overall, the ubiquity of these phages hints at their importance for plasmid ecology, and can provide opportunities in water quality monitoring and in ecological management of mobile resistance genes.

A novel and affordable bioaugmentation strategy with microbial extracts to accelerate the biodegradation of emerging contaminants in different media.

This study describes a new bioaugmentation alternative based on the application of aqueous aerated extracts from a biomixture acclimated with ibuprofen, diclofenac and triclosan. This bioaugmentation strategy was assayed in biopurification systems (BPS) and in contaminated aqueous solutions to accelerate the removal of these emerging contaminants. Sterilized extracts or extracts from the initial uncontaminated biomixture were used as controls. In BPS, the dissipation of 90% of diclofenac and triclosan required, respectively, 60 and 108 days less than in the controls. The metabolite methyl-triclosan was determined at levels 12 times lower than in controls. In the bioaugmented solutions, ibuprofen was almost completely eliminated (99%) in 21 days and its hydroxylated metabolites were also determined to be at lower levels than in the controls. The plasmidome of acclimated biomixtures and its extract appeared to maintain certain types of plasmids but degradation related genes became less evident. Several dominant OTUs found in the extract identified as Flavobacterium and Fluviicola of the phylum Bacteroidetes, Thermomicrobia (phylum Chloroflexi) and Nonomuraea (phylum Actinobacteria), may be responsible for the enhanced dissipation of these contaminants. This bioaugmentation strategy represents an advantageous tool to facilitate in situ bioaugmentation.

Influence of sewage sludge stabilization method on microbial community and the abundance of antibiotic resistance genes.

Municipal sewage sludge (MSS) and other biosolids are of high interest for agriculture. These nutrient-rich organic materials can potentially serve as organic fertilizers. Besides an increase of organic matter in soil, other positive effects were shown after their application. Especially the positive influence on circular economy increased the attention paid to management of MSS in recent years. Unfortunately, the use of sewage sludge has some drawbacks. Biosolids are frequently polluted with heavy metals, xenobiotic organic compounds and industrial chemicals, which may be hazardous for the environment and humans. Here, we investigated the influence of stabilization method and the size of wastewater treatment plant on the structure of microbial communities as well as the abundance of antibiotic resistance genes (ARG) and mobile genetic elements (MGE). All tested ARG and MGE were detectable in almost all of the samples. Interestingly, the presence of MGE as well as particular heavy metals correlated positively with the presence of several ARG. We conclude that the distribution of ARG and MGE in biosolids originated from municipal wastewater treatment plants, cannot be explained by the size of the facility or the applied stabilization method. Moreover, we postulate that the presence of pollutants and long-term impacts should be assessed prior to a possible use of sewage sludge as fertilizer.