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.

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.

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.

Bacterial community dynamics within an aerobic granular sludge reactor treating wastewater loaded with pharmaceuticals.

Pharmaceuticals are micropollutants often present in wastewater treatment systems. In this study, the potential impact of such micropollutants on the bacterial population within aerobic granular sludge (AGS) bioreactor was investigated. The AGS bacterial community structure and composition were accessed combining DGGE fingerprinting and barcoded pyrosequencing analysis. Both revealed the existence of a dynamic bacterial community, independently of the pharmaceuticals presence. The AGS microbiome at both phylum and class levels varied over time and, after stopping pharmaceuticals feeding, the bacterial community did not return to its initial composition. Nevertheless, most of the assigned OTUs were present throughout the different operational phases. This core microbiome, represented by over 72% of the total sequences in each phase, probably played an important role in biological removal processes, avoiding their failure during the disturbance period. Quantitative-PCR revealed that pharmaceuticals load led to gradual changes on the abundance of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB) and polyphosphate-accumulating organisms (PAO) but their persistence during that phase demonstrated the resilience of such bacterial groups. AGS microbiome changed over time but a core community was maintained, probably ensuring the accomplishment of the main biological removal processes.

Diversity of endophytic Pseudomonas in Halimione portulacoides from metal(loid)-polluted salt marshes.

Phytoremediation assisted by bacteria is seen as a promising alternative to reduce metal contamination in the environment. The main goal of this study was to characterize endophytic Pseudomonas isolated from Halimione portulacoides, a metal-accumulator plant, in salt marshes contaminated with metal(loid)s. Phylogenetic analysis based on 16S rRNA and gyrB genes showed that isolates affiliated with P. sabulinigri (n = 16), P. koreensis (n = 10), P. simiae (n = 5), P. seleniipraecipitans (n = 2), P. guineae (n = 2), P. migulae (n = 1), P. fragi (n = 1), P. xanthomarina (n = 1), and Pseudomonas sp. (n = 1). Most of these species have never been described as endophytic. The majority of the isolates were resistant to three or more metal(loid)s. Antibiotic resistance was frequent among the isolates but most likely related to species-intrinsic features. Common acquired antibiotic resistance genes and integrons were not detected. Plasmids were detected in 43.6 % of the isolates. Isolates that affiliated with different species shared the same plasmid profile but attempts to transfer metal resistance to receptor strains were not successful. Phosphate solubilization and IAA production were the most prevalent plant growth promoting traits, and 20 % of the isolates showed activity against phytopathogenic bacteria. Most isolates produced four or more extracellular enzymes. Preliminary results showed that two selected isolates promote Arabidopsis thaliana root elongation. Results highlight the diversity of endophytic Pseudomonas in H. portulacoides from contaminated sites and their potential to assist phytoremediation by acting as plant growth promoters and as environmental detoxifiers.

Intralesional Patterns of MRI ADC Maps Predict Outcome in Experimental Stroke.

BACKGROUND: After acute ischemia, the tissue that is at risk of infarction can be detected by perfusion-weighted imaging/diffusion-weighted imaging (PWI/DWI) mismatch but the time that is needed to process PWI limits its use. As DWI is highly sensitive to acute ischemic tissue damage, we hypothesized that different ADC patterns represent areas with a different potential for recovery. METHODS: In a model of permanent middle cerebral artery occlusion (pMCAO), Sprague-Dawley rats were randomly distributed to sham surgery and pMCAO. We further separated the pMCAO group according to intralesional ADC pattern (homogeneous or heterogeneous). At 24 h after ischemia induction, we analyzed lesion size, functional outcome, cell death expression, and brain protection markers including ROS enzyme NOX-4. MRI included DWI (ADC maps), DTI (tractography), and PWI (CBF, CBV and MTT). RESULTS: The lesion size was similar in pMCAO rats. Animals with a heterogeneous pattern in ADC maps showed better functional outcome in Rotarod test (p = 0.032), less expression of cell death (p = 0.014) and NOX-4 (p = 0.0063), higher intralesional CBF (p = 0.0026) and larger PWI/DWI mismatch (p = 0.007). CONCLUSIONS: In a rodent model for ischemic stroke, intralesional heterogeneity in ADC maps was related to better functional outcome in lesions of similar size and interval after pMCAO. DWI ADC maps may assist in the early identification of ischemic tissue with an increased potential for recovery as higher expression of acute protection markers, lower expression of cell death, increased PWI/DWI mismatch, and higher intralesional CBF were present in animals with a heterogeneous ADC pattern.

Contribution of chemical water properties to the differential responses of bacterioneuston and bacterioplankton to ultraviolet-B radiation.

The surface microlayer (SML) is characterized by different physicochemical properties from underlying waters (UW). However, whether these differences in abiotic factors underlie the distinct sensitivity of bacterioneuston (i.e. SML bacteria) and bacterioplankton to environmental stressors remains to be addressed. We investigated the contribution of abiotic factors to the UV-B sensitivity of bacterioneuston and bacterioplankton. Nutrients (especially nitrogen and phosphate) emerged as important determinants of bacterial UV-B sensitivity. The role of particles, nutrients, and dissolved organic components on bacterial UV-B sensitivity was further evaluated using dilution cultures. Filtered samples were twofold more UV sensitive than unfiltered samples, suggesting a UV-protective effect of particles. High nutrient concentrations attenuated bacterial UV-B sensitivity (up to 40%), compared with unamended conditions, by influencing bacterial physiology and/or community composition. Suspending cells in natural water, particularly from the SML, also attenuated UV-B sensitivity (up to 23%), compared with suspension in an artificial mineral solution. Bioassays using Pseudomonas sp. strain NT5I1.2B revealed that chemical water properties influence UV-induced oxidative damage. UV-B sensitivity was associated with high cell-specific activities. The chemical environment of the SML and UW influences UV-B effects on the corresponding bacterial communities. Maintaining low cell activities might be advantageous in stressful environments, like the SML.

Effects of UV radiation on the lipids and proteins of bacteria studied by mid-infrared spectroscopy.

Knowledge of the molecular effects of UV radiation (UVR) on bacteria can contribute to a better understanding of the environmental consequences of enhanced UV levels associated with global climate changes and will help to optimize UV-based disinfection strategies. In the present work, the effects of exposure to UVR in different spectral regions (UVC, 100-280 nm; UVB, 280-320 nm; and UVA, 320-400 nm) on the lipids and proteins of two bacterial strains ( Acinetobacter sp. strain PT5I1.2G and Pseudomonas sp. strain NT5I1.2B) with distinct UV sensitivities were studied by mid-infrared spectroscopy. Exposure to UVR caused an increase in methyl groups associated with lipids, lipid oxidation, and also led to alterations in lipid composition, which were confirmed by gas chromatography. Additionally, mid-infrared spectroscopy revealed the effects of UVR on protein conformation and protein composition, which were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), oxidative damage to amino acids, and changes in the propionylation, glycosylation and/or phosphorylation status of cell proteins. Differences in the targets of UVR in the two strains tested were identified and may explain their discrepant UV sensitivities. The significance of the results is discussed from an ecological standpoint and with respect to potential improvements in UV-based disinfection technologies.

Wavelength dependence of biological damage induced by UV radiation on bacteria.

The biological effects of UV radiation of different wavelengths (UVA, UVB and UVC) were assessed in nine bacterial isolates displaying different UV sensitivities. Biological effects (survival and activity) and molecular markers of oxidative stress [DNA strand breakage (DSB), generation of reactive oxygen species (ROS), oxidative damage to proteins and lipids, and the activity of antioxidant enzymes catalase and superoxide dismutase] were quantified and statistically analyzed in order to identify the major determinants of cell inactivation under the different spectral regions. Survival and activity followed a clear wavelength dependence, being highest under UVA and lowest under UVC. The generation of ROS, as well as protein and lipid oxidation, followed the same pattern. DNA damage (DSB) showed the inverse trend. Multiple stepwise regression analysis revealed that survival under UVA, UVB and UVC wavelengths was best explained by DSB, oxidative damage to lipids, and intracellular ROS levels, respectively.

Effects of UV-B radiation on the structural and physiological diversity of bacterioneuston and bacterioplankton.

The effects of UV radiation (UVR) on estuarine bacterioneuston and bacterioplankton were assessed in microcosm experiments. Bacterial abundance and DNA synthesis were more affected in bacterioplankton. Protein synthesis was more inhibited in bacterioneuston. Community analysis indicated that UVR has the potential to select resistant bacteria (e.g., Gammaproteobacteria), particularly abundant in bacterioneuston.

Evaluation of NucliSens-AmpliScreen methodology to detect subtypes G of HIV-1 and 4c/4d of HCV in the screening of blood donors.

BACKGROUND AND OBJECTIVES: Albeit, the NucliSens Extractor combined with the Ampliscreen was validated for application in NAT minipool screening, a study to evaluate the reliability of the procedure in relation to subtypes G of human immunodeficiency virus (HIV)-1 RNA and 4c/4d of hepatitis C virus (HCV) RNA should be performed, due to their genetic differences and the high frequency in our country. STUDY DESIGN: Samples from patients infected with subtypes G of HIV-1 RNA and 4c/4d of HCV RNA were diluted with negative plasma and tested eight times for each concentration. For nucleic acid extraction we used an automated silica-based extraction method (NucliSens Extractor) and for amplification and detection the AmpliScreen HIV-1 version 1.5 and AmpliScreen HCV version 2.0 (Roche Diagnostic Systems) were applied. RESULTS: The sensitivity for HIV-1 RNA genotype G using the NucliSens-AmpliScreen method-95% detection limit (95% CI) of 25 (18-50) copies per ml-is comparable with those described for genotypes B and E and to that obtained by the Multiprep procedure. In the case of HCV, the sensitivity of the method was also similar, when we compared the detection limits obtained for genotype 4c/4d-95% detection limit (95% CI) of 34 (24-71) IU/ml-with the genotype 1 published. CONCLUSIONS: The data presented here suggest that these infections will not be missed because of genetic variation, as the platform exhibited similar limits of detection for the subtypes evaluated, meeting the sensitivity requirements set by the regulatory bodies.