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Escherichia coli Ec36 was recovered from a patient in Portugal after treatment with meropenem and colistin. Besides an IncF plasmid with Tn1441d-blaKPC-3, already reported in clinical strains in this country, E. coli Ec36 co-harbored an IncX4::mcr-1 gene. Results highlight emerging co-resistance to carbapenems and polymyxins after therapy with drugs from both classes. [ABSTRACT FROM AUTHOR]

Many members of the genus Psychrobacter are endemic in extremely cold and saline environments and the genus has been described as only marginally successful in warmer habitats. In a previous study the Psychrobacter genus was, unexpectedly, the most frequently isolated bacterial genus from the sea-surface microlayer ( SML) and the underlying water ( UW) of a temperate estuary ( Ria de Aveiro, Portugal). Here we analysed the diversity in Psychrobacter populations inhabiting this estuary. Samples were collected at three dates and three locations from sea- SML and UW. Isolated Psychrobacter strains were well-adapted to temperatures and salt concentrations above the ones described as optimal for most members of this genus. Hydrocarbon-degrading potential was not confirmed for these strains. We developed and optimized a reliable and specific denaturing gradient gel electrophoresis ( DGGE)-based method for the analysis of Psychrobacter populations in aquatic systems. DGGE profiles inferred that Psychrobacter populations were very stable in the estuary, a strong indication for the presence of well-adapted phylotypes. The analysis of genus-specific clone libraries revealed a surprisingly high diversity among Psychrobacter in Ria de Aveiro. Results indicated that novel species were probably cultivated. Significant differences between sea- SML and UW Psychrobacter communities were revealed. Observed diversity trends may be related to environmental factors such as salinity and/or anthropogenic pressures such as contamination with hydrocarbons. [ABSTRACT FROM AUTHOR]

Background: Antibiotic-resistant bacteria have been surveyed widely in water bodies, but few studies have determined the diversity of antibiotic-resistant bacteria in river waters. This study was undertaken to investigate the origin of resistance among polluted river bacterial isolates in Tunisia. Methods: In this study 128 isolates resistant to β-lactam antibiotics were obtained from 2 polluted rivers in the north of Tunisia. Isolates were identified using Phoenix phenotyping criteria. The occurrence of blaTEM, blaSHV, blaCTX-M, blaCMY, blaVIM, and blaIMP was studied by polymerase chain reaction (PCR) amplification and sequencing, and the genetic relatedness of the 16 IMP-producing Klebsiella pneumoniae isolates was analyzed by comparison of XbaI pulsed-field gel electrophoresis (PFGE) profiles. Results: Using Phoenix phenotyping criteria, diverse genera of bacteria were identified with different rates of prevalence and with different minimum inhibitory concentrations against different antibiotics. The occurrence of blaTEM, blaSHV, blaCTXM, blaCMY, blaVIM, and blaIMP genes was confirmed. The DNA sequences upstream and downstream of blaIMP genes were determined, revealing that all IMP-encoding genes constituted the first cassette of class 1 integrons, followed by aacA gene cassettes encoding aminoglycoside resistance. Comparison of PFGE profiles showed that only 2 of the isolates were clonal, the other 14 displaying unique profiles. The blaCTX-M gene was the most dominant of the extended-spectrum β-lactamase (ESBL) genes, while the blaTEM gene was the second-most dominant. Conclusion: The discovery of highly diverse ESBL-producing bacteria and metallo-β-lactamases, particularly blaIMP, in polluted river water raises alarms with regard to the potential dissemination of antibiotic-resistant bacteria in communities through river environments. [ABSTRACT FROM AUTHOR]

The most common mechanism of resistance to β-lactam antibiotics is the production of β-lactamases. These enzymes are encoded by genes that evolve rapidly, thus constituting a group characterized by high levels of molecular diversity. Most of the genetic determinants of resistance to β-lactam antibiotics characterized until now were obtained from clinical isolates. This study was designed in order to exploit the presence of β-lactamase gene sequences in an aquatic environment, and to get information on the distinctive features of those sequences when compared to others available on databases. DNA sequences potentially encoding proteins of three different families of clinically relevant β-lactamases were assessed: TEM, IMP and OXA-2 derivatives. The presence of bla sequences in DNA extracted from water samples from the lagoon Ria de Aveiro was checked by PCR and hybridization. Sequences representing the three families of β-lactamases studied were detected. The molecular diversity of the amplicons was assessed by cloning and sequence analysis, and denaturing gradient gel electrophoresis (PCR-DGGE) separation. Most of the retrieved sequences (particularly sequences representing blaTEM and blaOXA-2) were identical or very similar to β-lactamase gene sequences previously characterized from clinical isolates. Phylogenetic analysis suggests that this aquatic ecosystem is a reservoir of molecular diverse putative bla sequences. The patterns of molecular diversity found within the β-lactamase gene families studied do not correspond to those reported in studies focussing on clinical isolates. [ABSTRACT FROM AUTHOR]

Abstract: The DNA sequence of the gyrB gene is a suitable phylogenetic marker for bacterial systematics. In this study, the diversity of Aeromonas spp. present in environmental samples was assessed by a PCR combined with DGGE approach. PCR primers targeting the gyrB gene of aeromonads were designed and the resulting amplicons were analyzed by DGGE. The gyrB-DGGE analysis was evaluated with Aeromonas isolates and reference strains allowing discrimination of the majority of strains. The gyrB-DGGE analysis is a powerful tool to monitor the presence and evaluate the diversity of aeromonads in complex samples, as is the case of water from a wastewater treatment plant. [Copyright &y& Elsevier]

The genus Brevibacterium includes pleomorphic Gram-positive bacteria with a high mol% G+C content. Species in the genus are difficult to identify by classical methods. The discriminatory power of DNA-based methods is assessed. Strains representing the four well established Brevibacterium species, and other related bacteria, were compared by amplified ribosomal DNA restriction analysis (ARDRA), repetitive-sequence-based PCR (rep-PCR) and ribotyping. Fingerprinting by rep-PCR and ribotyping provided complex genomic profiles with the highest discriminatory potential for molecular typing at the strain level, whereas ARDRA showed differentiation from the genus to the species levels. A high degree of heterogeneity within the genus Brevibacterium is apparent, thus indicating that the taxonomy of the genus should be further studied. [Copyright &y& Elsevier]

A potentially new thermotolerant B. licheniformis strain (code name I89), producer of an antibiotic active against Gram-positive bacteria, was genetically characterized and compared with the type strain B. licheniformis ATCC 10716, producer of bacitracin. Studies on DNA base composition (G + C content) and DNA reassociation revealed that the two strains show around 76% homology. Nevertheless, results obtained by rRNA hybridization, with a heterologous probe coding for most of the 16S region of the rRNA operon of Bacillus subtilis, revealed differences in the number of copies for that gene and in the hybridization pattern. Additionally, a different restriction digestion pattern was obtained when DNA was digested with the enzymes NotI, SmaI and analyzed by PFGE. The I89 strain holds a 7.6-kb plasmid not present in the reference strain. The existence of various unique restriction sites and also the stability of this plasmid make it ideal for the future development of a cloning and expression vector. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Antimicrobial resistance indicators were evaluated in a WWTP over 1 year. • Treatment and seasonality influenced the bacterial community and resistome. • intI1, bla GES and qacE∆1 genes correlated with the total concentration of ARGs. • bla GES and bla VIM genes correlated with the concentration of several ARGs' classes. • Integron-related genes are good indicators of antibiotic resistance in wastewater. 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, bla GES and qacE∆1 were the most strongly correlated with the total concentration of ARGs. Genes bla GES and bla VIM , 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. [Display omitted] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The dose-dependent variation of oxidative cellular damage imposed by UVB exposure in a representative estuarine bacterial strain, Pseudomonas sp. NT5I1.2B, was studied at different growth phases (mid-exponential, late-exponential, and stationary), growth temperatures (15 °C and 25 °C) and growth media (nutrient-rich Tryptic Soy Broth [TSB] and nutrient-poor M9). Survival and markers of oxidative damage (lipid peroxidation, protein carbonylation, DNA strand breakage, and DNA–protein cross-links) were monitored during exposure to increasing UVB doses (0–60 kJ m−2). Oxidative damage did not follow a clear linear dose-dependent pattern, particularly at high UVB doses (＞10 kJ m−2), suggesting a dynamic interaction between damage induction and repair during irradiation and/or saturation of oxidative damage. Survival of stationary phase cells generally exceeded that of exponential phase cells by up to 33.5 times; the latter displayed enhanced levels of DNA–protein cross-links (up to 15.6-fold) and protein carbonylation (up to 6.0-fold). Survival of mid-exponential phase cells was generally higher at 15 °C than at 25 °C (up to 6.6-fold), which was accompanied by lower levels of DNA strand breaks (up to 4000-fold), suggesting a temperature effect on reactive oxygen species (ROS) generation and/or ROS interaction with cellular targets. Survival under medium–high UVB doses (＞10 kJ m−2) was generally higher (up to 5.4-fold) in cells grown in TSB than in M9. These results highlight the influence of growth conditions preceding irradiation on the extent of oxidative damage induced by UVB exposure in bacteria. [ABSTRACT FROM AUTHOR]

Abstract: The present work aimed to identify the reactive oxygen species (ROS) produced during UV-B exposure and their biochemical targets, in a set of bacterial isolates displaying different UV susceptibilities. For that, specific exogenous ROS scavengers (catalase/CAT, superoxide dismutase/SOD, sodium azide and mannitol) were used. Biological effects were assessed from total bacterial number, colony counts and heterotrophic activity (glucose uptake and respiration). DNA strand breakage, ROS generation, oxidative damage to proteins and lipids were used as markers of oxidative stress. Sodium azide conferred a statistically significant protection in terms of lipid oxidation and cell survival, suggesting that singlet oxygen might play an important role in UV-B induced cell inactivation. Mannitol exerted a significant protection against DNA strand breakage and protein carbonylation, assigning hydroxyl radicals to DNA and protein damage. The addition of exogenous CAT and SOD significantly protected the capacity for glucose uptake and respiration, suggesting that superoxide and H2O2 are involved in the impairment of activity during UV-B exposure. The observation that amendment with ROS scavengers can sometimes also exert a pro-oxidant effect suggests that the intracellular oxidant status of the cell ultimately determines the efficiency of antioxidant defenses. [Copyright &y& Elsevier]

We compared the resistomes within polluted and unpolluted rivers, focusing on extended-spectrum beta-lactamase (ESBL) genes, in particular blaCTX-M. Twelve rivers from a Portuguese hydrographic basin were sampled. Physicochemical and microbiological parameters of water quality were determined, and the results showed that 9 rivers were classified as unpolluted (UP) and that 3 were classified as polluted (P). Of the 225 cefotaxime-resistant strains isolated, 39 were identified as ESBL-producing strains, with 18 carrying a blaCTX-M gene (15 from P and 3 from UP rivers). Analysis of CTX-M nucleotide sequences showed that 17 isolates produced CTX-M from group 1 (CTX-M-1, -3, -15, and -32) and 1 CTX-M that belonged to group 9 (CTX-M-14). A genetic environment study revealed the presence of different genetic elements previously described for clinical strains. ISEcp1 was found in the upstream regions of all isolates examined. Culture-independent blaCTX-M-like libraries were comprised of 16 CTX-M gene variants, with 14 types in the P library and 4 types in UP library, varying from 68% to 99% similarity between them. Besides the much lower level of diversity among CTX-M-like genes from UP sites, the majority were similar to chromosomal ESBLs such as blaRAHN-1. The results demonstrate that the occurrence and diversity of blaCTX-M genes are clearly different between polluted and unpolluted lotic ecosystems; these findings favor the hypothesis that natural environments are reservoirs of resistant bacteria and resistance genes, where anthropogenic-driven selective pressures may be contributing to the persistence and dissemination of genes usually relevant in clinical environments. [ABSTRACT FROM AUTHOR]

Abstract: In this study, the occurrence and diversity of integrons were evaluated in 697 isolates belonging to Enterobacteriaceae and Aeromonas spp. isolated from urban wastewaters. Screening of integrons was performed by dot blot hybridization and intI-positive strains were further characterized. The global prevalence of integrons was 3.73%. Three new gene cassettes were identified: a novel aadA variant (aadA17), a gene putatively involved in cell signaling (dcyA) and an open reading frame of unknown function interrupted by a novel insertion sequence (orfER.17::ISAs12). In total, thirteen different gene cassette arrays were detected, 4 representing novel integrons: intI1-dcyA-tniC, intI1-orfER.1.7::ISAs12-aadA13-qacEΔ1-sul1, intI1-aacA4-catB3-bla OxA-10-aadA1-qacEΔ1-sul1 and intI1-catB8-aadA17-qacEΔ1-sul1. Approximately 80% of strains were resistant to at least 3 antibiotics of different classes. The presence of novel integron structures in treated effluents suggests that domestic wastewaters may favor the formation of novel combinations of gene cassettes. Moreover, the high prevalence of multiresistant strains highlights the urgent need to employ effective means of effluent disinfection to avoid dissemination of antibiotic-resistant bacteria. [Copyright &y& Elsevier]

The use of biosolids as fertilizers in agriculture can lead to the exposure of soil biota to sulfidised silver nanoparticles (Ag 2 S 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 Ag 2 S NPs or AgNO 3 on the soil microbiome, using an indoor mesocosm. After 28 days of exposure, Ag 2 S 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 Ag 2 S NPs-treated soil toward the control. Nevertheless, stronger effects were observed in AgNO 3 -treated soil, over time, due to its higher silver dissolution rate in porewater. Additionally, only the AgNO 3 -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 Ag 2 S NPs on the soil microbiome composition, while AgNO 3 exerted a stronger effect in both composition and functional parameters. [Display omitted] • We studied Ag 2 S NPs effects on soil bacterial community in an indoor mesocosm. • Ag 2 S NPs change the community structure, at the class, genera and OTU level. • A significant increase of NOB was estimated in communities exposed to AgNO 3. • Variants of amoA and nxrB genes were altered, over-time, in silver-treated soils. [ABSTRACT FROM AUTHOR]

The toxicity of silver nanoparticles (AgNPs) in soil bacterial communities (SBC) has been widely reported. However, the bacterial related endpoints have not been effectively considered in the environmental risk assessment (ERA) for nanomaterials. Thus, we aimed to study the long-term effects of AgNPs, or AgNO 3 (counterpart), [5 μg (Ag) kg−1] on SBC: (1) at structural level (using denaturing gradient gel electrophoresis-DGGE) and, (2) in terms of bacterial induced tolerance (which is the foundation of the pollution-induced community tolerance - PICT) using a disc diffusion assay (0.1 μg, 1 μg or 10 μg of each silver form). After day-56 of exposure, the structure of SBC was affected by AgNPs and AgNO 3 , though still sharing 71.9% similarity with the non-exposed SBC. Also, the SBC homogeneity significantly decreased after exposure to silver, suggesting a possible tolerance effect. Regarding tolerance assays, AgNO 3 was more effective in inhibiting the SBC growth than AgNPs, and both in a dose-dependent manner (10 μg > 1 μg > 0.1 μg). A second exposure revealed a decrease in tolerance of SBC to AgNO 3. Also, a lower amount of AgNO 3 is needed to change SBC tolerance. PICT analysis revealed that previous exposure to AgNPs did not increase tolerance-effect in SBC. Overall, AgNPs changes the SBC structure and tolerance, but does not increase the tolerance on a second exposure. Thus, this study highlights the usefulness of combined bacterial endpoints (DGGE, and PICT-disc diffusion tests) for potential inclusion in the ERA of AgNPs in soil ecosystems. [Display omitted] • Silver nanoparticles (AgNPs) had effects on soil bacterial community (SBC). • AgNPs changed the SBC in terms of structure and tolerance. • No increase in tolerance of SBC was detected in a second exposure. • Bacterial endpoints support environmental risk assessment of AgNPs in soil. [ABSTRACT FROM AUTHOR]

Abstract: The fingerprinting methods referred to as MSP-PCR (microsatellite-primed polymerase chain reaction) and rep-PCR (repetitive-sequence-based polymerase chain reaction) were used to discriminate between species of Botryosphaeriaceae (Fungi, Ascomycota). Several primers were tested with both methods and each primer enabled clear differentiation of all the species tested. Cluster analysis of banding patterns of the isolates corresponded well with known species delineations based on morphology and phylogenetic analysis. The methods described in this paper provide simple and rapid procedures that can be used for routine differentiation of Botryosphaeriaceae isolates at the species level. The two methods are also useful for studying intraspecific variability. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Background: Coronavirus disease 2019 (COVID-19) has placed a tremendous strain on healthcare services. This study, prepared by a large international panel of stroke experts, assesses the rapidly growing research and personal experience with COVID-19 stroke and offers recommendations for stroke management in this challenging new setting: modifications needed for prehospital emergency rescue and hyperacute care; inpatient intensive or stroke units; posthospitalization rehabilitation; follow-up including at-risk family and community; and multispecialty departmental developments in the allied professions. Summary: The severe acute respiratory syndrome coronavirus 2 uses spike proteins binding to tissue angiotensin-converting enzyme (ACE)-2 receptors, most often through the respiratory system by virus inhalation and thence to other susceptible organ systems, leading to COVID-19. Clinicians facing the many etiologies for stroke have been sobered by the unusual incidence of combined etiologies and presentations, prominent among them are vasculitis, cardiomyopathy, hypercoagulable state, and endothelial dysfunction. International standards of acute stroke management remain in force, but COVID-19 adds the burdens of personal protections for the patient, rescue, and hospital staff and for some even into the postdischarge phase. For pending COVID-19 determination and also for those shown to be COVID-19 affected, strict infection control is needed at all times to reduce spread of infection and to protect healthcare staff, using the wealth of well-described methods. For COVID-19 patients with stroke, thrombolysis and thrombectomy should be continued, and the usual early management of hypertension applies, save that recent work suggests continuing ACE inhibitors and ARBs. Prothrombotic states, some acute and severe, encourage prophylactic LMWH unless bleeding risk is high. COVID-19-related cardiomyopathy adds risk of cardioembolic stroke, where heparin or warfarin may be preferable, with experience accumulating with DOACs. As ever, arteritis can prove a difficult diagnosis, especially if not obvious on the acute angiogram done for clot extraction. This field is under rapid development and may generate management recommendations which are as yet unsettled, even undiscovered. Beyond the acute management phase, COVID-19-related stroke also forces rehabilitation services to use protective precautions. As with all stroke patients, health workers should be aware of symptoms of depression, anxiety, insomnia, and/or distress developing in their patients and caregivers. Postdischarge outpatient care currently includes continued secondary prevention measures. Although hoping a COVID-19 stroke patient can be considered cured of the virus, those concerned for contact safety can take comfort in the increasing use of telemedicine, which is itself a growing source of patient-physician contacts. Many online resources are available to patients and physicians. Like prior challenges, stroke care teams will also overcome this one. Key Messages: Evidence-based stroke management should continue to be provided throughout the patient care journey, while strict infection control measures are enforced. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is a health challenge across human, animal and environmental settings. In the environment, metals may contribute to antibiotic resistance selection. This study aimed to investigate the role of copper and zinc in the selection of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in a riverine bacterial community. Using a microcosm approach, bacteria in water samples were exposed to 50 μg L−1 and 100 μg L−1 of copper and zinc, for 20 days. The prevalence of ARB was determined from colony forming units counts in media with and without antibiotics. A significant increase in the prevalence of cefotaxime-resistant (from 2.3% in control to 9.5% in Cu50 and 16.8% in Cu100) and tetracycline-resistant bacteria (from 0.03% to 0.23% in Cu100) was observed in communities exposed to copper. Zinc exposure resulted in an increase in the prevalence of cefotaxime-resistant bacteria (from 24.6% to 91.3% in Zn50 and 72.4% in Zn100) and of kanamycin-resistant bacteria (from 6.1% to 24.1% in Zn50 and 43% in Zn100). Cefotaxime- and kanamycin-resistant bacteria belonged to genera intrinsically resistant to these compounds. DGGE profiling confirmed that metal exposure altered the structure and diversity of bacterial communities. Changes in the abundance of genes usually associated with mobile genetic elements (bla CTX-M , bla TEM , tet (A) and intI1) were not detected after exposure. Results demonstrated the selection of bacteria intrinsically resistant to antibiotics imposed by copper and zinc exposure, suggesting an important role played by cross-resistance mechanisms. • Microcosms were used to study metal effects on antibiotic resistance selection. • Cu exposure increased cefotaxime- and tetracycline-resistant bacteria prevalence. • Zn exposure increased cefotaxime- and kanamycin-resistant bacteria prevalence. • Metal exposure selected for bacteria intrinsically resistant to antibiotics. • Abundance of acquired genes (tet(A) and intI1) was not affected by metal exposure. [ABSTRACT FROM AUTHOR]

The increasing number of Chromobacterium haemolyticum human infection reports, especially in tropical regions and connected with environmental sources, resulted in an urge to better describe this species. This study aimed to characterize the C. haemolyticum resistome, virulence determinants and genetic platforms related with genome plasticity. A comparative genomic analysis was conducted between clinical C. haemolyticum genomes publicly available and the genome of an environmental isolate obtained in this study. The pangenome of C. haemolyticum was calculated and a total of 3378 core genes were predicted in its core genome, corresponding to 51.7% of the pangenome. Genetic determinants putatively encoding resistance to beta-lactams, fosfomycin, aminoglycosides and trimethoprim were predicted in all genomes, possibly constituting the intrinsic resistome of this species. In terms of resistance to beta-lactams, 4 genes were predicted encoding beta-lactamases of classes A, C and D. Moreover, the analysis of Chromobacterium genomes and C. haemolyticum environmental isolates reinforced the role of this genus as progenitor of the blaKPC gene. Putative virulence factors (VFs) were predicted in all genomes, related to adherence, toxins production, colonization and cell invasion. Secretion systems, including type III, were detected. A significant number of transposases and genomic islands were predicted in C. haemolyticum, in some cases above the average reported for Gram-negative bacterial genomes. We conclude that C. haemolyticum strains, including those of environmental origin, present a noteworthy collection of antibiotic resistance genes and VFs. Furthermore, sequences related to gene mobility and genome plasticity suggest high adaptability potential and a possible role as disseminator of antibiotic resistance. [ABSTRACT FROM AUTHOR]

• Essential oils (EOs) were obtained for the first time from aromatic plant byproducts. • Solvent-free microwave extraction was applied to obtain EOs from byproducts. • Thirteen volatile compounds were quantified in EOs of S. montana by GC–MS. • Carvacrol was the most abundant component (825–950 μg/mg) of S. montana EOs. • S. montana EOs exhibited antimicrobial activity against poultry pathogenic bacteria. Satureja montana is a worldwide consumed aromatic plant whose essential oils (EOs) are used as spice and preservative by food industry. Only the leaves are marketed, generating a large amount of stems as byproducts. It is possible that the EOs present in these byproducts represent a source of compounds with antimicrobial activity, as observed for the leaves. In this work, dried S. montana byproducts were used to extract EOs. Although differences were observed for the harvest year (0.42 ± 0.11 mg/g in 2016 and 0.14 ± 0.07 mg/g in 2017), monoterpenoids were the major components, accounting for 84.4–97.6%, being carvacrol the most abundant component (825–950 μg/mg). Sesquiterpenoids accounted for 0.3 to 0.5%. Similar EOs yield and composition were obtained using solvent-free microwave extraction (16 min using 100 g of S. montana stems, previously, soaked in water and drained) or hydrodistillation (120 min using 1000 mL of water/ 100 g of dried S. montana stems). The in vitro minimal inhibitory concentrations (MICs) of S. montana EOs against Escherichia coli ATCC 25922, Salmonella enterica sv Anatum SF2, and Staphylococcus aureus ATCC 6538, which belong to pathogenic species with economic impact in poultry industry, were 225 μg/mL, 250 μg/mL, and 150 μg/mL, respectively. As these MICs are similar to the ones reported for carvacrol against the same or related strains, it can be concluded that carvacrol is the active compound in S. montana byproducts. [ABSTRACT FROM AUTHOR]

Upon consumption, dietary nitrate is reduced to nitrite in the oral cavity and to nitric oxide (•NO) in the stomach. Here, •NO increases mucosal blood flow, mucus thickness and prevents microbial infections. However, the impact of nitrate on gut microbiota, a pleiotropic organism essential to maintain gastrointestinal and systemic welfare, remains elusive. This study investigates the impact of nitrate on gut microbiota profile and ensued mucosal effects during dysbiosis. Male Wistar rats were randomly distributed in 4 groups and the drinking water was supplemented for 7 days as follows: 1) antibiotic cocktail (neomycin, bacitracin and imipenem), 2) antibiotic cocktail + sodium nitrate, 3) sodium nitrate and 4) regular drinking water. Animals were weighted daily and feces were collected before and after the treatment. The stomach was isolated and the expression of occludin, claudin-5 as well as myeloperoxidase and iNOS was studied. Bacterial DNA was analyzed in fecal samples by PCR-DGGE genetic fingerprinting. Nitrate prevented antibiotic-induced body weight loss (1.9 ± 1.8% vs 8.9 ± 1.8%, p < 0.05) and cecamegalia (7.1 ± 0.5% vs 5.6 ± 0.4%, p < 0.05). Gastric expression of occludin and claudin-5 tended to decrease during dysbiosis but both protein levels were recovered following nitrate consumption (p < 0.05). Similarly, nitrate inhibited the overexpression of myeloperoxidase and iNOS observed under dysbiosis (p < 0.05). Broad spectrum antibiotics significantly decreased microbiota richness and diversity in comparison to controls (p = 0.0016). After 7 days of treatment, whereas antibiotics reduced microbiota richness by 56%, it was observed that nitrate was able to prevent such microbial loss to only 48%, although without statistical differences (p = 0.068). This data suggests that dietary nitrate may be envisaged as a key component of functional foods with beneficial impact on gastric mucosal integrity during antibiotherapy but further studies are mandatory to better ascertain as to whether it modulates intestinal microbiota in terms of taxonomic and functional levels. Image 1 • Nitrate prevents body weight loss and cecamegalia induced by antibiotics. • Gastric occludin and claudin-5 are recovered by nitrate under dysbiosis. • Nitrate lowers iNOS and MPO expression under antibiotic-induced dysbiosis. • Nitrate tends to increase microbiota richness but without significant differences. [ABSTRACT FROM AUTHOR]

Abstract Oxytetracycline (OTC) is a broad-spectrum antibiotic widely used in livestock production. Like many other pharmaceuticals, OTC is not completely metabolized by the organism and thus, increasing amounts of the compound are being detected in the aquatic environment. The assessment of the environmental risk of pharmaceuticals is hindered by their very low concentrations and specific modes of action and thus relevant exposure scenarios and sensitive endpoints are needed. Thus, this work aimed to study the long-term effect of OTC exposure in zebrafish (at behavior and biochemical levels) and associated bacterial communities (fish gut and water bacterial communities). Results revealed that at behavioral level, boldness increase (manifested by increased exploratory behavior of a new environment) was observed in fish exposed to low OTC concentrations. Moreover, changes in fish swimming pattern were observed in light periods (increased stress response: hyperactivity and freezing) probably due to photo-sensibility conferred by OTC exposure. Effects at biochemical level suggest that long-term exposure to OTC interfere with cellular energy allocation mainly by reducing lipids levels and increasing energy consumption. Moreover, evidences of oxidative damage were also observed (reduced levels of TG, GST and CAT). The analysis of water and gut microbiome revealed changes in the structure and diversity of bacterial communities potentially leading to changes in communities' biological function. Some of the effects were observed at the lowest concentration tested, 0.1 μg/L which is a concentration already detected in the environment and thus clearly demonstrating the need of a serious ecotoxicological assessment of OTC effects on non-target organisms. Graphical abstract Image 1 Highlights • Zebrafish exposed to oxytetracycline present different feeding behavior pattern. • Oxytetracycline induced fish boldness and hyperactivity in light periods. • Energetic reserves and oxidative stress enzymes decrease after long-term exposure. • Bacterial communities of fish gut and exposure water were affected by oxytetracycline. [ABSTRACT FROM AUTHOR]

Disinfection by UV radiation is one of the most promising solutions to reduce the bacterial load and antibiotic resistance in the final effluents of urban wastewater treatment plants (UWTP). Our aim was to evaluate the fate of cefotaxime-resistant Enterobacteriaceae and Extended Spectrum Beta-Lactamase (ESBL) producers in a full-scale system that includes UV-C disinfection. Over treatment, the abundance of cefotaxime-resistant Enterobacteriaceae was reduced, with reductions of 1.9 log units after secondary treatment (STW samples) and 1.8 log following UV disinfection (UTW samples). These reductions, did not reflect the variations in the prevalence of cefotaxime-resistant Enterobacteriaceae , estimated to be of 3% in raw wastewater (RW), 18% in STW and 3% in UTW. A significant increase of cefotaxime-resistant bacterial counts (0.5 log; p  < 0.05) was observed after 3 days of storage. In a total of 1799 cefotaxime-resistant Enterobacteriaceae isolates, 15% harboured bla CTX-M ( n  = 274), 11% bla TEM ( n  = 194) and 4% bla SHV ( n  = 72). While the ESBL gene prevalence decreased over treatment, the prevalence of the intI 1 gene decreased after ST but slightly increased in UTW samples. The bla CTX-M -carriers were identified as Escherichia coli and Klebsiella pneumoniae , mostly multi-drug resistant (90.5%) and carrying integrase genes (82.8%). The bla CTX-M gene variants (48 bla CTX-M-15 , 9 bla CTX-M-32, 8 bla CTX-M-1, 5 bla CTX-M-27, and 2 bla CTX-M-14 ) were flanked by IS Ecp1 , IS Ecp 1/IS 26 , IS 903 and ORF477 in 8 different arrangements. The IncF plasmid replicon type was highly prevalent among bla CTX-M -carrying Escherichia coli (74.5%) while IncR predominated among K. pneumoniae (54.5%). Our results confirmed the potential of UV-C disinfection to remove antibiotic resistant bacteria. Still, resistant Enterobacteriaceae (about 30 × 10 6  cells per m 3 of water), presenting traits that might potentiate antibiotic resistance spread, are released in the final effluent. In addition, a significant regrowth was observed after storage. These results suggest that improvements of wastewater disinfection are still required to minimize the risks associated with UWTP discharges. [ABSTRACT FROM AUTHOR]

Urban wastewater treatment plants (UWTPs) are reservoirs of antibiotic resistance. Wastewater treatment changes the bacterial community and inevitably impacts the fate of antibiotic resistant bacteria and antibiotic resistance genes (ARGs). Some bacterial groups are major carriers of ARGs and hence, their elimination during wastewater treatment may contribute to increasing resistance removal efficiency. This study, conducted at a full-scale UWTP, evaluated variations in the bacterial community and ARGs loads and explored possible associations among them. With that aim, the bacterial community composition (16S rRNA gene Illumina sequencing) and ARGs abundance (real-time PCR) were characterized in samples of raw wastewater (RWW), secondary effluent (sTWW), after UV disinfection (tTWW), and after a period of 3 days storage to monitoring possible bacterial regrowth (tTWW-RE). Culturable enterobacteria were also enumerated. Secondary treatment was associated with the most dramatic bacterial community variations and coincided with reductions of ~2 log-units in the ARGs abundance. In contrast, no significant changes in the bacterial community composition and ARGs abundance were observed after UV disinfection of sTWW. Nevertheless, after UV treatment, viability losses were indicated ~2 log-units reductions of culturable enterobacteria. The analysed ARGs ( qnrS , bla CTX-M , bla OXA-A , bla TEM , bla SHV , sul1 , sul2 , and intI1 ) were strongly correlated with taxa more abundant in RWW than in the other types of water, and which associated with humans and animals, such as members of the families Campylobacteraceae , Comamonadaceae , Aeromonadaceae , Moraxellaceae, and Bacteroidaceae . Further knowledge of the dynamics of the bacterial community during wastewater treatment and its relationship with ARGs variations may contribute with information useful for wastewater treatment optimization, aiming at a more effective resistance control. [ABSTRACT FROM AUTHOR]

Pine Wilt Disease (PWD) has a significant impact on Eurasia pine forests. The microbiome of the nematode (the primary cause of the disease), its insect vector, and the host tree may be relevant for the disease mechanism. The aim of this study was to characterize these microbiomes, from three PWD-affected areas in Portugal, using Denaturing Gradient Gel Electrophoresis, 16S rRNA gene pyrosequencing, and a functional inference-based approach (PICRUSt). The bacterial community structure of the nematode was significantly different from the infected trees but closely related to the insect vector, supporting the hypothesis that the nematode microbiome might be in part inherited from the insect. Sampling location influenced mostly the tree microbiome (P < 0.05). Genes related both with plant growth promotion and phytopathogenicity were predicted for the tree microbiome. Xenobiotic degradation functions were predicted in the nematode and insect microbiomes. Phytotoxin biosynthesis was also predicted for the nematode microbiome, supporting the theory of a direct contribution of the microbiome to tree-wilting. This is the first study that simultaneously characterized the nematode, tree and insect-vector microbiomes from the same affected areas, and overall the results support the hypothesis that the PWD microbiome plays an important role in the disease's development. [ABSTRACT FROM AUTHOR]