Integron-associated genes are reliable indicators of antibiotic resistance in wastewater despite treatment- and seasonality-driven fluctuations.

The present study aims to characterize the bacterial community, resistome and integron abundance of a municipal wastewater treatment plant (WWTP) over the course of 12 months and evaluate the year-long performance of integron-related genes as potential indicators of antibiotic resistance mechanisms in influents and effluents. For that, total DNA was extracted and subjected to 16S rRNA-targeted metabarcoding, high-throughput (HT) qPCR (48 targets) and standard qPCR (5 targets). Targets included integrase genes, antibiotic resistance genes (ARGs) and putative pathogenic groups. A total of 16 physicochemical parameters determined in the wastewater samples were also considered. Results revealed that the WWTP treatment significantly impacted the bacterial community, as well as the content in ARGs and integrase genes. Indeed, there was a relative enrichment from influent to effluent of 13 pathogenic groups (e.g., Legionella and Mycobacterium) and genes conferring resistance to sulphonamides, aminoglycosides and disinfectants. Effluent samples (n = 25) also presented seasonal differences, with an increase of the total ARGs' concentration in summer, and differences between winter and summer on relative abundance of sulphonamide and disinfectant resistance mechanisms. From the eight putative integron-related genes selected, all were positively correlated with the total ARGs' content in wastewater and the relative abundance of resistance to most of the specific antibiotic classes. The genes intI1, blaGES and qacE∆1 were the most strongly correlated with the total concentration of ARGs. Genes blaGES and blaVIM, were better correlated to resistance to beta-lactams, aminoglycosides and tetracyclines. This study supports the use of integron-related genes as powerful indicators of antibiotic resistance in wastewater, being robust despite the variability caused by wastewater treatment and seasonality.

Responses of soil microbiome to copper-based materials (nano and bulk) for agricultural applications: An indoor-mesocosm experiment.

The foreseen increasing application of copper-based nanomaterials (Cu-NMs), replacing or complementing existing Cu-agrochemicals, may negatively impact the soil microbiome. Thus, we studied the effects on soil microbiome function and composition of nano copper oxide (nCuO) or copper hydroxide NMs in a commercial (Kocide®3000) or a lab-synthetized formulation (nCu(OH)2) or bulk copper hydroxide (Cu(OH)2-B), at the commonly recommended Cu dose of 50 mg(Cu)kg-1 soil. Microbial responses were studied over 28 days in a designed indoor mesocosm. On day-28, in comparison to non-treated soil (CT), all Cu-treatments led to a reduction in dehydrogenase (95% to 68%), arylsulfatase (41% to 27%), and urease (40% to 20%) activity. There was a 32% increase in the utilization of carbon substrates in the nCuO-treatment and an increased abundance of viable bacteria in the nCu(OH)2-treatment (75% of heterotrophic and 69% of P-solubilizing bacteria). The relative abundance of Acidobacteria [Kocide®3000, nCuO, and Cu(OH)2-B treatments] and Flavobacteriia [nCu(OH)2-treatment] was negatively affected by Cu exposure. The abundance of Cu-tolerant bacteria increased in soils treated with Kocide®3000 (Clostridia) and nCu(OH)2 (Gemmatimonadetes). All Cu-treated soils exhibited a reduced abundance of denitrification-related genes (0.05% of nosZ gene). The DTPA-extractable pool of ionic Cu(II) varied among treatments: Cu(OH)2-B > Kocide®3000 âˆ¼ nCuO>nCu(OH)2, which may explain changes on the soil microbiome composition, at the genera and OTU levels. Thus, our study revealed that Cu-materials (nano and bulk) influence the soil microbiome with implications on its ecological role. It highlights the importance of assessing the impact of Cu-materials under dynamic and complex exposure scenarios and emphasizes the need for specific regulatory frameworks for NMs.

Hidden threats in the plastisphere: Carbapenemase-producing Enterobacterales colonizing microplastics in river water.

Carbapenem resistance poses a significant burden on healthcare systems worldwide. Microplastics (MPs) have emerged as potential contributors to antibiotic resistance spread in the environment. However, the link between MPs and carbapenem resistance remains unexplored. We investigated the prevalence of carbapenem-resistant bacteria colonizing MPs placed in a river. Three replicates of a mixture of polypropylene (PP), polyethylene (PE) and polyethylene terephthalate (PET) and of PET alone were placed both upstream and downstream a wastewater treatment plant (WWTP) discharge. Carbapenem-resistant Enterobacterales (CRE) were further characterized by phenotypic tests and whole-genome sequencing. The abundance of carbapenem-resistant bacteria on MPs increased significantly downstream the WWTP. Their prevalence was higher in the MPs mixture compared to PET alone. CRE strains colonizing MPs included Klebsiella pneumoniae (n = 3), Klebsiella quasipneumoniae (n = 3), Raoultella ornithinolytica (n = 2), Enterobacter kobei (n = 1) and Citrobacter freundii (n = 1), most (n = 8) recovered after the WWTP discharge. All strains exhibited at least one of the tested virulence traits (biofilm formation at 37 °C, haemolytic activity and siderophore production), were multi-drug resistant and carried carbapenemase-encoding genes [blaKPC-3 (n = 5), blaGES-5 (n = 2) or blaKPC-3 + blaGES-5 (n = 3)]. Uncommon phenotypes of resistance to imipenem/relebactam (n = 3) and ceftazidime/avibactam (n = 2) were observed. Two blaKPC-3-positive K. pneumoniae successfully transfer this gene trough conjugation. Genome analysis predicted all strains as human pathogens. The blaKPC-3 was associated with the Tn4401d transposon on a pBK30683-like plasmid in most of the isolates (n = 7). The blaGES-5 was mostly linked to class 3 integrons. A K. pneumoniae strain belonging to the outbreak-causing high-risk clone ST15 carried both blaKPC-3 and blaCTX-M-15. Two K. quasipneumoniae isolates carried the plasmid-mediated colistin resistance gene mcr-9. Our results underscore the role of MPs as vectors for CRE dissemination, particularly following WWTPs discharges. MPs may act as carriers, facilitating the dissemination of carbapenemase-encoding genes and potentially contributing to increased CRE incidence in the environment.

Structural and Functional Shifts in the Microbial Community of a Heavy Metal-Contaminated Soil Exposed to Short-Term Changes in Air Temperature, Soil Moisture and UV Radiation.

The interplay between metal contamination and climate change may exacerbate the negative impact on the soil microbiome and, consequently, on soil health and ecosystem services. We assessed the response of the microbial community of a heavy metal-contaminated soil when exposed to short-term (48 h) variations in air temperature, soil humidity or ultraviolet (UV) radiation in the absence and presence of Enchytraeus crypticus (soil invertebrate). Each of the climate scenarios simulated significantly altered at least one of the microbial parameters measured. Irrespective of the presence or absence of invertebrates, the effects were particularly marked upon exposure to increased air temperature and alterations in soil moisture levels (drought and flood scenarios). The observed effects can be partly explained by significant alterations in soil properties such as pH, dissolved organic carbon, and water-extractable heavy metals, which were observed for all scenarios in comparison to standard conditions. The occurrence of invertebrates mitigated some of the impacts observed on the soil microbial community, particularly in bacterial abundance, richness, diversity, and metabolic activity. Our findings emphasize the importance of considering the interplay between climate change, anthropogenic pressures, and soil biotic components to assess the impact of climate change on terrestrial ecosystems and to develop and implement effective management strategies.

Microplastics accumulate priority antibiotic-resistant pathogens: Evidence from the riverine plastisphere.

Microplastics (MPs) might accumulate and transport antibiotic-resistant bacteria (ARB) in aquatic systems. We determined the abundance and diversity of culturable ciprofloxacin- and cefotaxime-resistant bacteria in biofilms covering MPs placed in river water, and characterized priority pathogens from these biofilms. Our results showed that the abundance of ARB colonizing MPs tends to be higher compared to sand particles. Also, higher numbers were cultivated from a mixture of polypropylene (PP), polyethylene (PE) and polyethylene terephthalate (PET), compared to PP and PET alone. Aeromonas and Pseudomonas isolates were the most frequently retrieved from MPs placed before a WWTP discharge while Enterobacteriaceae dominated the culturable plastisphere 200 m after the WWTP discharge. Ciprofloxacin- and/or cefotaxime-resistant Enterobacteriaceae (n = 54 unique isolates) were identified as Escherichia coli (n = 37), Klebsiella pneumoniae (n = 3), Citrobacter spp. (n = 9), Enterobacter spp. (n = 4) and Shigella sp. (n = 1). All isolates presented at least one of the virulence features tested (i.e. biofilm formation, haemolytic activity and production of siderophores), 70% carried the intI1 gene and 85% exhibited a multi-drug resistance phenotype. Plasmid-mediated quinolone resistance genes were detected in ciprofloxacin-resistant Enterobacteriaceae [aacA4-cr (40% of the isolates), qnrS (30%), qnrB (25%), and qnrVC (8%)], along with mutations in gyrA (70%) and parC (72%). Cefotaxime-resistant strains (n = 23) harbored blaCTX-M (70%), blaTEM (61%) and blaSHV (39%). Among CTX-M producers, high-risk clones of E. coli (e.g. ST10 or ST131) and K. pneumoniae (ST17) were identified, most of which carrying blaCTX-M-15. Ten out of 16 CTX-M producers were able to transfer blaCTX-M to a recipient strain. Our results demonstrated the occurrence of multidrug resistant Enterobacteriaceae in the riverine plastisphere, harboring ARGs of clinical concern and exhibiting virulence traits, suggesting a contribution of MPs to the dissemination of antibiotic-resistant priority pathogens. The type of MPs and especially water contamination (e.g. by WWTPs discharges) seem to determine the resistome of the riverine plastisphere.

Bioremediation of coastal aquaculture effluents spiked with florfenicol using microalgae-based granular sludge - a promising solution for recirculating aquaculture systems.

Aquaculture is a crucial industry in the agri-food sector, but it is linked to serious environmental problems. There is a need for efficient treatment systems that allow water recirculation to mitigate pollution and water scarcity. This work aimed to evaluate the self-granulation process of a microalgae-based consortium and its capacity to bioremediate coastal aquaculture streams that sporadically contain the antibiotic florfenicol (FF). A photo-sequencing batch reactor was inoculated with an autochthonous phototrophic microbial consortium and was fed with wastewater mimicking coastal aquaculture streams. A rapid granulation process occurred within ca. 21 days, accompanied by a substantially increase of extracellular polymeric substances in the biomass. The developed microalgae-based granules exhibited high and stable organic carbon removal (83-100%). Sporadically wastewater contained FF which was partially removed (ca. 5.5-11.4%) from the effluent. In periods of FF load, the ammonium removal slightly decreased (from 100 to ca. 70%), recovering 2 days after FF feeding ceased. A high-chemical quality effluent was obtained, complying with ammonium, nitrite, and nitrate concentrations for water recirculation within a coastal aquaculture farm, even during FF feeding periods. Members belonging to the Chloroidium genus were predominant in the reactor inoculum (ca. 99%) but were replaced from day-22 onwards by an unidentified microalga from the phylum Chlorophyta (>61%). A bacterial community proliferated in the granules after reactor inoculation, whose composition varied in response to feeding conditions. Bacteria from the Muricauda and Filomicrobium genera, Rhizobiaceae, Balneolaceae, and Parvularculaceae families, thrived upon FF feeding. This study demonstrates the robustness of microalgae-based granular systems for aquaculture effluent bioremediation, even during periods of FF loading, highlighting their potential as a feasible and compact solution in recirculation aquaculture systems.

Report and Comparative Genomics of an NDM-5-Producing Escherichia coli in a Portuguese Hospital: Complex Class 1 Integrons as Important Players in blaNDM Spread.

BACKGROUND: New Delhi metallo-beta-lactamase (NDM) has been spreading across the globe, but the causes of its success are poorly understood. We characterized a blaNDM-5-positive Escherichia coli strain from a Portuguese hospital and conducted comparative genomic analyses to understand the role of clonal background and horizontal gene transfer in blaNDM-5 dissemination. METHODS: After blaNDM PCR screening and genome sequencing, Ec355340 was subjected to mating, transformation, and plasmid curing assays and MICs determination for several antibiotics. Comparison with data compiled from public databases was performed. RESULTS: blaNDM-5 was in a complex integron co-located in a FIB-FII plasmid (pEc355340_NDM-5). The mating assays were unsuccessful, but plasmid transformation into a susceptible host led to resistance to all beta-lactams and to sulfamethoxazole-trimethoprim. The profile of virulence genes (n = 73) was compatible with extraintestinal pathogenesis. An analysis of genomes from public databases suggested that blaNDM-5 has rarely been associated with ST156 strains (such as Ec355340), while is has frequently been found on strains of the ST10 clonal complex. However, ST156 may play a role in the co-spreading of blaNDM and mcr genes. Regardless, comparative genomics confirmed the presence of blaNDM in similar complex integrons in plasmids (48/100 plasmids most similar to pEc355340_NDM-5) and ST156 genomes (20/41 blaNDM-positive genomes). CONCLUSIONS: blaNDM-5 and other blaNDM variants were more frequently associated to complex integrons than previously reported and, therefore, these platforms may be important drivers in their dissemination. The identification of blaNDM-5 for the first time in Portugal could be a game-changer in the current Portuguese antibiotic resistance scenario, as this gene encodes a higher-level resistance phenotype, and its spread may be facilitated due to the association with complex integrons.

CTX-M-Producing Bacteria Isolated from a Highly Polluted River System in Portugal.

Enterobacteriaceae resistant to third-generation cephalosporins are a great concern for public health, as these are first-line drugs to treat infections. The production of carbapenemases and extended spectrum beta-lactamases (ESBLs) and/or the overexpression of AmpC ß-lactamases are the main mechanisms of resistance to these antibiotics. Among the ESBLs, CTX-M ß-lactamases are the most prevalent worldwide. Our aims were to determine the prevalence of cefotaxime-resistant Enterobacteriaceae along a heavily polluted river and characterize blaCTX-M carriers. River water was collected in 11 sites along the main course and tributaries, in two sampling moments. Water quality was evaluated and a collection of cefotaxime-resistant isolates was obtained. blaCTX-M carriers were characterized regarding phylogenetic affiliation, clonality, antibiotic susceptibility, gene diversity, and context. Water presented very low quality in all sites. From 147 cefotaxime-resistant isolates, 46% carried blaCTX-M and were affiliated with Escherichia, Klebsiella, Enterobacter, and Citrobacter. Molecular typing revealed clonal isolates in different sites and over the two years, suggesting survival of the strains in the river or continuous pollution inputs from the same sources. Eight variants of blaCTX-M were found, with blaCTX-M-15 being the most prevalent (52.5%). Sites with a lower water quality showed the highest resistance rates and prevalence of blaCTX-M, suggesting that river water may embody human health risks.

Common and distinctive genomic features of Klebsiella pneumoniae thriving in the natural environment or in clinical settings.

The Klebsiella pneumoniae complex is comprised of ubiquitous bacteria that can be found in soils, plants or water, and as humans' opportunistic pathogens. This study aimed at inferring common and distinctive features in clinical and environmental K. pneumoniae. Whole genome sequences of members of the K. pneumoniae complex (including K. variicola, n = 6; and K. quasipneumoniae, n = 7), of clinical (n = 78) and environmental (n = 61) origin from 21 countries were accessed from the GenBank. These genomes were compared based on phylogeny, pangenome and selected clinically relevant traits. Phylogenetic analysis based on 2704 genes of the core genome showed close relatedness between clinical and environmental strains, in agreement with the multi-locus sequence typing. Eight out of the 62 sequence types (STs) identified, included both clinical and environmental genomes (ST11, ST14, ST15, ST37, ST45, ST147, ST348, ST437). Pangenome-wide association studies did not evidence significant differences between clinical and environmental genomes. However, the genomes of clinical isolates presented significantly more exclusive genes related to antibiotic resistance/plasmids, while the environmental isolates yielded significantly higher allelic diversity of genes related with functions such as efflux or oxidative stress. The study suggests that K. pneumoniae can circulate among the natural environment and clinical settings, probably under distinct adaptation pressures.

Short-Term Responses of Soil Microbial Communities to Changes in Air Temperature, Soil Moisture and UV Radiation.

We analyzed the effects on a soil microbial community of short-term alterations in air temperature, soil moisture and ultraviolet radiation and assessed the role of invertebrates (species Enchytraeus crypticus) in modulating the community's response to these factors. The reference soil, Lufa 2.2, was incubated for 48 h, with and without invertebrates, under the following conditions: standard (20 °C + 50% water holding capacity (WHC)); increased air temperature (15-25 °C or 20-30 °C + 50% WHC); flood (20 °C + 75% WHC); drought (20 °C + 25% WHC); and ultraviolet radiation (UV) (20 °C + 50% WHC + UV). BIOLOG EcoPlates and 16S rDNA sequencing (Illumina) were used to assess the microbial community's physiological profile and the bacterial community's structure, respectively. The bacterial abundance (estimated by 16S rDNA qPCR) did not change. Most of the conditions led to an increase in microbial activity and a decrease in diversity. The structure of the bacterial community was particularly affected by higher air temperatures (20-30 °C, without E. crypticus) and floods (with E. crypticus). Effects were observed at the class, genera and OTU levels. The presence of invertebrates mostly resulted in the attenuation of the observed effects, highlighting the importance of considering microbiome-invertebrate interactions. Considering future climate changes, the effects described here raise concern. This study provides fundamental knowledge to develop effective strategies to mitigate these negative outcomes. However, long-term studies integrating biotic and abiotic factors are needed.

KPC-3-, GES-5-, and VIM-1-Producing Enterobacterales Isolated from Urban Ponds.

Carbapenems are antibiotics of pivotal importance in human medicine, the efficacy of which is threatened by the increasing prevalence of carbapenem-resistant Enterobacterales (CRE). Urban ponds may be reservoirs of CRE, although this hypothesis has been poorly explored. We assessed the proportion of CRE in urban ponds over a one-year period and retrieved 23 isolates. These were submitted to BOX-PCR, PFGE, 16S rDNA sequencing, antibiotic susceptibility tests, detection of carbapenemase-encoding genes, and conjugation assays. Isolates were affiliated with Klebsiella (n = 1), Raoultella (n = 11), Citrobacter (n = 8), and Enterobacter (n = 3). Carbapenemase-encoding genes were detected in 21 isolates: blaKPC (n = 20), blaGES-5 (n = 6), and blaVIM (n = 1), with 7 isolates carrying two carbapenemase genes. Clonal isolates were collected from different ponds and in different campaigns. Citrobacter F6, Raoultella N9, and Enterobacter N10 were predicted as pathogens from whole-genome sequence analysis, which also revealed the presence of several resistance genes and mobile genetic elements. We found that blaKPC-3 was located on Tn4401b (Citrobacter F6 and Enterobacter N10) or Tn4401d (Raoultella N9). The former was part of an IncFIA-FII pBK30683-like plasmid. In addition, blaGES-5 was in a class 3 integron, either chromosomal (Raoultella N9) or plasmidic (Enterobacter N10). Our findings confirmed the role of urban ponds as reservoirs and dispersal sites for CRE.

Microbial Associations of Abyssal Gorgonians and Anemones (>4,000 m Depth) at the Clarion-Clipperton Fracture Zone.

Deep coral-dominated communities play paramount roles in benthic environments by increasing their complexity and biodiversity. Coral-associated microbes are crucial to maintain fitness and homeostasis at the holobiont level. However, deep-sea coral biology and their associated microbiomes remain largely understudied, and less from remote and abyssal environments such as those in the Clarion-Clipperton Fracture Zone (CCZ) in the tropical Northeast (NE) Pacific Ocean. Here, we study microbial-associated communities of abyssal gorgonian corals and anemones (>4,000 m depth) in the CCZ; an area harboring the largest known global reserve of polymetallic nodules that are commercially interesting for the deep-sea nodule mining. Coral samples (n = 25) belonged to Isididae and Primnoidae families, while anemones (n = 4) to Actinostolidae family. Significant differences in bacterial community compositions were obtained between these three families, despite sharing similar habitats. Anemones harbored bacterial microbiomes composed mainly of Hyphomicrobiaceae, Parvibaculales, and Pelagibius members. Core microbiomes of corals were mainly dominated by different Spongiibacteraceae and Terasakiellaceae bacterial members, depending on corals' taxonomy. Moreover, the predicted functional profiling suggests that deep-sea corals harbor bacterial communities that allow obtaining additional energy due to the scarce availability of nutrients. This study presents the first report of microbiomes associated with abyssal gorgonians and anemones and will serve as baseline data and crucial insights to evaluate and provide guidance on the impacts of deep-sea mining on these key abyssal communities.

Food Ingredients Derived from Lemongrass Byproduct Hydrodistillation: Essential Oil, Hydrolate, and Decoction.

Essential oil (EO), hydrolate, and nondistilled aqueous phase (decoction) obtained from the hydrodistillation of lemongrass byproducts were studied in terms of their potential as food ingredients under a circular economy. The EO (0.21%, dry weight basis) was composed mainly of monoterpenoids (61%), the majority being citral (1.09 g/kg). The minimal inhibitory concentrations (MIC) of lemongrass EO against Escherichia coli, Salmonella enterica, and Staphylococcus aureus, were 617, 1550, and 250 µg/mL, respectively. This effect was dependent on the citral content. Particularly for Gram-negative bacteria, a synergism between citral and the remaining EO compounds enhanced the antimicrobial activity. The polymeric material obtained from the nondistilled aqueous phase was composed of phenolic compounds (25% gallic acid equivalents) and carbohydrates (22%), mainly glucose (66 mol%). This polymeric material showed high antioxidant activity due to bound phenolic compounds, allowing its application as a functional dietary fiber ingredient. Matcha green tea formulations were successfully mixed with lemongrass hydrolate containing 0.21% EO (dry weight basis) with 58% of monoterpenoids, being citral at 0.73 g/kg, minimizing matcha astringency with a citrus flavor and extending the product shelf life. This holistic approach to essential oils' hydrodistillation of Cymbopogon citratus byproducts allows for valorizing of the essential oil, hydrolate, and decoction for use as food ingredients.

Occurrence and distribution of Carbapenem-resistant Enterobacterales and carbapenemase genes along a highly polluted hydrographic basin.

We determined the distribution and temporal variation of Carbapenem Resistant Enterobacterales (CRE), carbapenemase-encoding genes and other antibiotic resistance genes (ARGs) in a highly polluted river (Lis River; Portugal), also assessing the potential influence of water quality to this distribution. Water samples were collected in two sampling campaigns performed one year apart (2018/2019) from fifteen sites and water quality was analyzed. CRE were isolated and characterized. The abundance of four ARGs (blaNDM, blaKPC, tetA, blaCTX-M), two Microbial Source Tracking (MST) indicators (HF183 and Pig-2-Bac) and the class 1 integrase gene (IntI1) was measured by qPCR. RESULTS: confirmed the poor quality of the Lis River water, particularly in sites near pig farms. A collection of 23 CRE was obtained: Klebsiella (n = 19), Enterobacter (n = 2) and Raoultella (n = 2). PFGE analysis revealed a clonal relationship between isolates obtained in different sampling years and sites. All CRE isolates exhibited multidrug resistance profiles. Klebsiella and Raoultella isolates carried blaKPC while Enterobacter harbored blaNDM. Conjugation experiments were successful for only four Klebsiella isolates. All ARGs were detected by qPCR on both sampling campaigns. An increase in ARGs and IntI1 abundances was detected in sites located downstream of wastewater treatment plants. Strong correlations were observed between blaCTX-M, IntI1 and the human-pollution marker HF183, and also between tetA and the pig-pollution marker Pig-2-bac, suggesting that both human- and animal-derived pollution in the Lis River are a potential source of ARGs. Plus, water quality parameters related to eutrophication and land use were significantly correlated with ARGs abundances. Our findings demonstrated that the Lis River encloses high levels of antibiotic resistant bacteria and ARGs, including CRE and carbapenemase-encoding genes. Overall, this study provides a better understanding on the impacts of water pollution resulting from human and animal activities on the resistome of natural aquatic systems.

Third generation cephalosporin-resistant Klebsiella pneumoniae thriving in patients and in wastewater: what do they have in common?

BACKGROUND: Klebsiella pneumoniae are ubiquitous bacteria and recognized multidrug-resistant opportunistic pathogens that can be released into the environment, mainly through sewage, where they can survive even after wastewater treatment. A major question is if once released into wastewater, the selection of lineages missing clinically-relevant traits may occur. Wastewater (n = 25) and clinical (n = 34) 3rd generation cephalosporin-resistant K. pneumoniae isolates were compared based on phenotypic, genotypic and genomic analyses. RESULTS: Clinical and wastewater isolates were indistinguishable based on phenotypic and genotypic characterization. The analysis of whole genome sequences of 22 isolates showed that antibiotic and metal resistance or virulence genes, were associated with mobile genetic elements, mostly transposons, insertion sequences or integrative and conjugative elements. These features were variable among isolates, according to the respective genetic lineage rather than the origin. CONCLUSIONS: It is suggested that once acquired, clinically relevant features of K. pneumoniae may be preserved in wastewater, even after treatment. This evidence highlights the high capacity of K. pneumoniae for spreading through wastewater, enhancing the risks of transmission back to humans.

New insights into the role of constitutive bacterial rhizobiome and phenolic compounds in two Pinus spp. with contrasting susceptibility to pine pitch canker.

The rhizobiome is being increasingly acknowledged as a key player in plant health and breeding strategies. The pine pitch canker (PPC), caused by the fungus Fusarium circinatum, affects pine species with varying susceptibility degrees. Our aims were to explore the bacterial rhizobiome of a susceptible (Pinus radiata) and a resistant (Pinus pinea) species together with other physiological traits, and to analyze shifts upon F. circinatum inoculation. Pinus seedlings were stem inoculated with F. circinatum spores and needle gas exchange and antioxidant-related parameters were analyzed in non-inoculated and inoculated plants. Rhizobiome structure was evaluated through 16S rRNA gene massive parallel sequencing. Species (non-inoculated plants) harbored distinct rhizobiomes (<40% similarity), where P. pinea displayed a rhizobiome with increased abundance of taxa described in suppressive soils, displaying plant growth promoting (PGP) traits and/or anti-fungal activity. Plants of this species also displayed higher levels of phenolic compounds. F. circinatum induced slight changes in the rhizobiome of both species and a negative impact in photosynthetic-related parameters in P. radiata. We concluded that the rhizobiome of each pine species is distinct and higher abundance of bacterial taxa associated to disease protection was registered for the PPC-resistant species. Furthermore, differences in the rhizobiome are paralleled by a distinct content in phenolic compounds, which are also linked to plants' resistance against PPC. This study unveils a species-specific rhizobiome and provides insights to exploit the rhizobiome for plant selection in nurseries and for rhizobiome-based plant-growth-promoting strategies, boosting environmentally friendly disease control strategies.

The impact of silver sulfide nanoparticles and silver ions in soil microbiome.

The use of biosolids as fertilizers in agriculture can lead to the exposure of soil biota to sulfidised silver nanoparticles (Ag2S NPs), generated during the wastewater treatment procedures. Considering the crucial role of microorganisms on soil functions, we aimed to study the effects of 10 mg kg-1 soil of Ag2S NPs or AgNO3 on the soil microbiome, using an indoor mesocosm. After 28 days of exposure, Ag2S NPs induced a significant change in the soil microbiome structure, at class, genera and OTU levels. For instance, a significantly higher abundance of Chitinophagia, known for its lignocellulose-degrading activity, was observed in Ag2S NPs-treated soil toward the control. Nevertheless, stronger effects were observed in AgNO3-treated soil, over time, due to its higher silver dissolution rate in porewater. Additionally, only the AgNO3-treated soil stimulates the abundance of ammonia-oxidizing (AOB; amoA gene) and nitrite-oxidizing (NOB; nxrB gene) bacteria, which are involved in the nitrification process. Distinct variants of amoA and nxrB genes emerged in silver-treated soils, suggesting a potential succession of AOB and NOB with different degree of silver-tolerance. Our study highlights the latter effects of Ag2S NPs on the soil microbiome composition, while AgNO3 exerted a stronger effect in both composition and functional parameters.

Gut and faecal bacterial community of the terrestrial isopod Porcellionides pruinosus: potential use for monitoring exposure scenarios.

This work aimed to characterize the gut and faeces bacterial communities (BC) of Porcellionides pruinosus using high-throughput sequencing. Isopods were collected from the field and kept in laboratory conditions similar to those normally applied in ecotoxicology tests. Faeces and purged guts of isopods (n = 3 × 30) were analysed by pyrosequencing the V3-V4 region of 16 S rRNA encoding gene. Results showed that gut and faecal BCs were dominated by Proteobacteria, particularly by an OTU (Operational Taxonomic Unit) affiliated to genus Coxiella. Diversity and richness values were statistically higher for faecal BC, mainly due to the occurrence of several low-abundance phylotypes. These results may reflect faecal carriage of bacterial groups that cannot settle in the gut. BCs of P. pruinosus comprised: (1) common members of the soil microbiota, (2) bacterial symbionts, (3) bacteria related to host metabolic/ecological features, and (4) bacterial etiological agents. Comparison of BC of this isopod species with the BC from other invertebrates revealed common bacterial groups across taxa. The baseline information provided by this work will assist the design and data interpretation of future ecotoxicological or biomonitoring assays where the analysis of P. pruinosus BC should be included as an additional indicator. CAPSULE: Terrestrial isopods bacterial communities might support ecotoxicological assays and biomonitoring processes as a valuable tool.

Effects of Long-Term Exposure to Increased Salinity on the Amphibian Skin Bacterium Erwinia toletana.

Amphibian's skin bacterial community may help them to cope with several types of environmental perturbations, including osmotic stress caused by increased salinity. This work assessed whether an amphibian skin bacterium could increase its tolerance to NaCl after a long-term exposure to this salt. A strain of Erwinia toletana, isolated from the skin of Pelophylax perezi, was exposed to two salinity scenarios (with 18 g/L of NaCl): (1) long-term exposure (for 46 days; Et-NaCl), and (2) long-term exposure followed by a recovery period (exposure for 30 days to NaCl and then to LB medium for 16 days; Et-R). After exposure, the sensitivity of E. toletana clonal populations to NaCl was assessed by exposing them to 6 NaCl concentrations (LB medium spiked with NaCl) plus a control (LB medium). Genotypic alterations were assessed by PCR-based molecular typing method (BOX-PCR). The results showed that tolerance of E. toletana to NaCl slightly increased after the long-term exposure, EC50 for growth were: 22.5 g/L (8.64-36.4) for Et-LB; 30.3 g/L (23.2-37.4) for Et-NaCl; and 26.1 g/L (19.332.9) for Et-R. Differences in metabolic activity were observed between Et-LB and Et-R and between Et-NaCl and Et-R, suggesting the use of different substrates by this bacterium when exposed to salinized environments. NaCl-induced genotypic alterations were not detected. This work suggests that E. toletana exposed to low levels of salinity, activate different metabolic pathways to cope with osmotic stress. These findings may be further explored to be used in bioaugmentation procedures through the supplementation with this bacterium of the skin microbiome of natural populations of amphibians exposed to salinization.

Tetracycline-Resistant Bacteria Selected from Water and Zebrafish after Antibiotic Exposure.

The emergence of antibiotic-resistant pathogens due to worldwide antibiotic use is raising concern in several settings, including aquaculture. In this work, the selection of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) was evaluated after exposure of zebrafish to oxytetracycline (OTC) for two months, followed by a recovery period. The selection of ARB in water and fish was determined using selective media. The abundance of tetA genes was estimated through qPCR. Higher prevalence of ARB was measured in all samples exposed to the antibiotic when compared to control samples, although statistical significance was only achieved five days after exposure. Isolates recovered from samples exposed to the antibiotic were affiliated with Pseudomonas and Stenotrophomonas. Various antibiotic susceptibility profiles were detected and 37% of the isolates displayed multidrug resistance (MDR). The selection of the tetA gene was confirmed by qPCR at the highest OTC concentration tested. Two MDR isolates, tested using zebrafish embryos, caused significant mortality, indicating a potential impact on fish health and survival. Overall, our work highlights the potential impact of antibiotic contamination in the selection of potential pathogenic ARB and ARGS.