The environment is considered a major reservoir of antimicrobial resistant microorganisms (AMR) and antimicrobial resistance genes (ARG). Colistin, a “last resort” antibiotic, is used for the treatment of severe infections caused by multidrug-resistant Gram-negative bacteria. The global dissemination of mobile colistin resistance genes (mcr) in natural and non-natural environments is a major setback in the fight against antimicrobial resistance. Hitherto, there is a limited number of studies screening this resistance determinant in bacteria from wildlife. In this study, we describe for the first time the detection of plasmid-mediated colistin resistance in Escherichia coli from wild ungulates in Portugal, which are also widely distributed across Europe. This information is critical to identify the importance of ungulates in the dissemination of resistant bacteria, and their corresponding genes, across the environment. Here, 151 resistant-Enterobacteriaceae isolated from 181 samples collected from different wild ungulate species throughout Portugal were screened for mcr genes. Four mcr-1-positive Escherichia coli were detected from four fallow deer individuals that were sampled in the same hunting ground. These four isolates harboured mcr-1-related IncP plasmids belonging to sequencing types ST155, ST533 and ST345 (n = 2), suggesting bacterial and/or plasmid circulation. All mcr-1-positive E. coli also showed other resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. All mcr-1-positive E. coli show additional resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. Our findings are upsetting, highlighting the global dissemination of colistin resistance genes in the whole ecosystem, which, under the One Health framework, emphasizes the urgent need for effective implementation of AMR surveillance and control in the human-animal-environment interfaces.

Antibiotic resistance is exacerbated by the exchange of antibiotic resistance genes (ARGs) between microbes from diverse habitats. Plasmids are important ARGs mobile elements and are spread by horizontal gene transfer (HGT). In this study, we demonstrated the presence of multi-resistant plasmids from inhalable particulate matter (PM) and its effect on gene horizontal transfer. Three transferable multi-resistant plasmids were identified from PM in a hospital, using conjugative mating assays and nanopore sequencing. pTAir-3 contained 26 horizontal transfer elements and 10 ARGs. Importantly pTAir-5 harbored carbapenem resistance gene (blaOXA) which shows homology to plasmids from human and pig commensal bacteria, thus indicating that PM is a media for antibiotic resistant plasmid spread. In addition, 125 μg/mL PM₂.₅ and PM₁₀ significantly increased the conjugative transfer rate by 110% and 30%, respectively, and augmented reactive oxygen species (ROS) levels. Underlying mechanisms were revealed by identifying the upregulated expressional levels of genes related to ROS, SOS, cell membranes, pilus generation, and transposition via genome-wide RNA sequencing. The study highlights the airborne spread of multi-resistant plasmids and the impact of inhalable PM on the horizontal transfer of antibiotic resistance.

Epidemiological studies have demonstrated that the general population’s exposure to bisphenol A (BPA) substitutes is ubiquitous. Bisphenol F (BPF), one of the main BPA substitutes, is increasingly replacing BPA in plastics for food and beverage applications. Accumulating evidence suggests that BPA exposure is associated with nonalcoholic fatty liver disease (NAFLD)-like changes. However, the potential effects of BPF on lipid homeostasis remain poorly understood. In the present study, an epidemiological analysis with LC-MS-MS revealed that the BPF concentrations in the serum of NAFLD patients were significantly higher than those in a control group. Supporting this result, using Oil Red O, BODIPY 493/503, LipidTox Deep Red staining and gas chromatography-time-of-flight mass spectrometry (TOF-MS) assays, we found that BPF exposure induced NAFLD-like changes, with obvious lipid droplet deposition, triglyceride (TG) and fatty acids increase in mouse livers. Meanwhile, lipid droplet deposition and TG increase induced by BPF were also observed in HepG2 cells, accompanied by autophagic flux blockade, including autophagosome accumulation and the decreased degradation of SQSTM1/p62. Using adenoviruses dual-reporter plasmid RFP-GFP-LC3, RFP-GFP-PLIN2 transfection, AO staining, and EGFR degradation assays, we demonstrated that BPF treatment impaired lysosomal degradative capacity, since BPF treatment obviously impaired lysosomal acidification, manifested as decreased lysosomal hydrolase cathepsin L (CTSL) and mature cathepsin D (CTSD) in HepG2 and mouse liver issues. Additionally, v-ATPase D, a multi-subunit enzyme that mediates acidification of eukaryotic intracellular organelles, significantly decreased after BPF exposure in both the vitro and in vivo studies.This study ascertained a novel mechanism involving dysfunctional of lysosomal degradative capacity induced by BPF, which contributes to lipophagic disorders and causes lipid droplet deposition. This work provides evidence that lysosomes may be a target organelle where BPF exerts its potential toxicity; therefore, novel intervention strategies targeting lysosome are promising for BPF-induced NAFLD-like changes.

The high global consumption of ibuprofen and its limited elimination by wastewater treatment plants (WWTPs), has led to the contamination of aquatic systems by this common analgesic and its metabolites. The potentially negative environmental and public health effects of this emerging contaminant have raised concerns, driving the demand for treatment technologies. The implementation of bacteria which mineralize organic contaminants in biopurification systems used to decontaminate water or directly in processes in WWTPs, is a cheap and sustainable means for complete elimination before release into the environment. In this work, an ibuprofen-mineralizing bacterial strain isolated from sediments of the River Elbe was characterized and assayed to remediate different ibuprofen-polluted media. Strain RW412, which was identified as Sphingopyxis granuli, has a 4.48 Mb genome which includes plasmid sequences which harbor the ipf genes that encode the first steps of ibuprofen mineralization. Here, we confirm that these genes encode enzymes which initiate CoA ligation to ibuprofen, followed by aromatic ring activation by a dioxygenase and retroaldol cleavage to unequivocally produce 4-isobutylcatechol and propionyl-CoA which then undergo further degradation. In liquid mineral salts medium, the strain eliminated more than 2 mM ibuprofen within 74 h with a generation time of 16 h. Upon inoculation into biopurification systems, it eliminated repeated doses of ibuprofen within a few days. Furthermore, in these systems the presence of RW412 avoided the accumulation of ibuprofen metabolites. In ibuprofen-spiked effluent from a municipal WWTP, ibuprofen removal by this strain was 7 times faster than by the indigenous microbiota. These results suggest that this strain can persist and remain active under environmentally relevant conditions, and may be a useful innovation to eliminate this emerging contaminant from urban wastewater treatment systems.

Horizontal gene transfer (HGT) plays an important role in the dissemination of antibiotic resistance genes. In sewer systems, human-associated and environmental bacteria are mixed together and exposed to many substances known to increase HGT, including various antibacterial compounds. In wastewaters, those substances are most often detected below concentrations known to induce HGT individually. Still, it is possible that such wastewaters induce HGT, for example via mixture effects. Here, a panel of antibiotics, biocides and other pharmaceuticals was measured in filter-sterilized municipal and hospital wastewater samples from Gothenburg, Sweden. The effects on HGT of the chemical mixtures in these samples were investigated by exposing a complex bacterial donor community together with a GFP-tagged E. coli recipient strain. Recipients that captured sulfonamide resistance-conferring mobile genetic elements (MGEs) from the bacterial community were enumerated and characterized by replicon typing, antibiotic susceptibility testing and long read sequencing. While exposure to municipal wastewater did not result in any detectable change in HGT rates, exposure to hospital wastewater was associated with an increase in the proportion of recipients that acquired sulfonamide resistance but also a drastic decrease in the total number of recipients. Although, concentrations were generally higher in hospital than municipal wastewater, none of the measured substances could individually explain the observed effects of hospital wastewater. The great majority of the MGEs captured were IncN plasmids, and resistance to several antibiotics was co-transferred in most cases. Taken together, the data show no evidence that chemicals present in the studied municipal wastewater induce HGT. Still, the increased relative abundance of transconjugants after exposure to hospital wastewater could have implications for the risks of both emergence and transmission of resistant bacteria.

Dibenzofuran (DBF) derivatives have caused serious environmental problems, especially those produced by paper pulp bleaching and incineration processes. Prominent for its resilient mutagenicity and toxicity, DBF poses a major challenge to human health. In the present study, a new strain of Pseudomonas aeruginosa, FA-HZ1, with high DBF-degrading activity was isolated and identified. The determined optimum conditions for cell growth of strain FA-HZ1 were a temperature of 30 °C, pH 5.0, rotation rate of 200 rpm and 0.1 mM DBF as a carbon source. The biochemical and physiological features as well as usage of different carbon sources by FA-HZ1 were studied. The new strain was positive for arginine double hydrolase, gelatinase and citric acid, while it was negative for urease and lysine decarboxylase. It could utilize citric acid as its sole carbon source, but was negative for indole and H2S production. Intermediates of DBF 1,2-dihydroxy-1,2-dihydrodibenzofuran, 1,2-dihydroxydibenzofuran, 2-hydroxy-4-(3′-oxo-3′H-benzofuran-2′-yliden)but-2-enoic acid, 2,3-dihydroxybenzofuran, 2-oxo-2-(2′-hydrophenyl)lactic acid, and 2-hydroxy-2-(2′-hydroxyphenyl)acetic acid were detected and identified through liquid chromatography-mass analyses. FA-HZ1 metabolizes DBF by both the angular and lateral dioxygenation pathways. The genomic study identified 158 genes that were involved in the catabolism of aromatic compounds. To identify the key genes responsible for DBF degradation, a proteomic study was performed. A total of 1459 proteins were identified in strain FA-HZ1, of which 100 were up-regulated and 104 were down-regulated. A novel enzyme “HZ6359 dioxygenase”, was amplified and expressed in pET-28a in E. coli BL21(DE3). The recombinant plasmid was successfully constructed, and was used for further experiments to verify its function. In addition, the strain FA-HZ1 can also degrade halogenated analogues such as 2, 8-dibromo dibenzofuran and 4-(4-bromophenyl) dibenzofuran. Undoubtedly, the isolation and characterization of new strain and the designed pathways is significant, as it could lead to the development of cost-effective and alternative remediation strategies. The degradation pathway of DBF by P. aeruginosa FA-HZ1 is a promising tool of biotechnological and environmental significance.

The increase of CO₂ concentration in the atmosphere, water and soil environment can lead to the changes in microbial activities. However, the transformation of antibiotic resistance genes has not been investigated in the presence of higher levels of CO₂. This study demonstrated that CO₂ facilitated the transformation of pUC19 plasmid, carrying ampicillin resistance genes, into Escherichia coli. Mechanism studies revealed that the type Ⅱ secretion system, type Ⅳ pilus and some other secretion systems were enhanced by CO₂, leading to DNA capture by pilus, larger cell pore sizes and more cell membrane channels. CO₂ also increased reactive oxygen species production, leading to SOS response and cell membrane damage. Besides, changes in intracellular Fe²⁺ and Mg²⁺ concentrations induced by CO₂ caused greater damage to the cell membrane and enhanced secretion systems, respectively. Overall, increased CO₂ provided more cell membrane channels for plasmid uptake and led to higher transformation efficiencies. The potential risk of a natural factor on the transformation of ARGs was first studied in this study, which helps us understand the fate of ARGs in ecosystems. As the carbon emission will continue to grow and enhance the enrichment of CO₂ in water and soil, the findings revealed a more severe public health issue under the background of carbon emission and CO₂ leakage.

Microbial source tracking (MST) DNA-based assays have been used to successfully solve fecal pollution problems in many countries, particularly in developed nations. However, their application in developing countries has been limited but continues to increase. In this study, sixteen endpoint and quantitative PCR (qPCR) assays targeting universal and human-, swine-, and cattle-specific Bacteroidales gene markers were modified for endpoint PCR, evaluated for their performance with sewage and fecal samples from the Tha Chin watershed and subsequently validated with samples from the Chao Phraya watershed, Thailand. Sample sizes of 81 composite samples (from over 1620 individual samples) of farm animals of each type as well as 19 human sewage samples from the Tha Chin watershed were calculated using a stratified random sampling design to achieve a 90% confidence interval and an expected prevalence (i.e., desired assay's sensitivity) of 0.80. The best universal and human-, swine-, and cattle-specific fecal markers were BacUni EP, HF183/BFDrev EP, Pig-2-Bac EP, and Bac3 assays, respectively. The detection limits for these assays ranged from 30 to 3000 plasmid copies per PCR. The positive predictive values were high in universal and swine- and cattle-specific markers (85–100%), while the positive predictive value of the human-specific assay was 52.2%. The negative predictive values in all assays were relatively high (90.8–100%). A suite of PCR assays in Thailand was established for potential MST use in environmental waters, which supports the worldwide applicability of Bacteroidales gene markers. This study also emphasizes the importance of using a proper sample size in assessing the performance of MST markers in a new geographic region.

Particulate air pollution being recognized to be responsible for short and long term health effects, regulations for particulate matter with an aerodynamic diameter less than 2.5 (PM2.5) are more and more restrictive. PM2.5 regulation is based on mass without taking into account PM2.5 composition that drives toxicity. Measurement of the oxidative potential (OP) of PM could be an additional PM indicator that would encompass the PM components involved in oxidative stress, the main mechanism of PM toxicity. We compared different methods to evaluate the intrinsic oxidative potential of PM2.5 sampled in Paris and their ability to reflect the oxidative and inflammatory response in bronchial epithelial cells used as relevant target organ cells. The dithiothreitol depletion assay, the antioxidant (ascorbic acid and glutathione) depletion assay (OPAO), the plasmid scission assay and the dichlorofluorescein (DCFH) oxidation assay used to characterize the OP of PM2.5 (10–100 μg/mL) provided positive results of different magnitude with all the PM2.5 samples used with significant correlation with different metals such as Cu and Zn as well as total polyaromatic hydrocarbons and the soluble organic fraction. The OPAO assay showed the best correlation with the production of intracellular reactive oxygen species by NCI-H292 cell line assessed by DCFH oxidation and with the expression of anti-oxidant genes (superoxide dismutase 2, heme-oxygenase-1) as well as the proinflammatory response (Interleukin 6) when exposed from 1 to 10 μg/cm2. The OPAO assay appears as the most prone to predict the biological effect driven by PM2.5 and related to oxidative stress.

Coastal waters are confluences receiving large amounts of point and non-point sources of pollution. An attempt was made to explore microbial community interactions in response to carbon, nitrogen and metal pollution. Additionally, experiments were designed to analyze the influence of these factors on horizontal gene transfer (HGT). Shift in bacterial diversity dynamics by arsenic stress and nutrient addition in coastal waters was explored by metagenomics of microcosm setups. Phylogenetic analysis revealed equal distribution of Gammaproteobacteria (29%) and Betaproteobacteria (28%) in control microcosm. This proportional diversity from control switched to unique distribution of Gammaproteobacteria (44.5%)> Flavobacteria (17.7%)> Bacteriodia (11.92%)> Betaproteobacteria (11.52%) in microcosm supplemented with carbon, nitrogen and metal (C + N + M). Among metal-stressed systems, alpha diversity analysis indicated highest diversity of genera in C + N + M followed by N + M > C+M> metal alone. Arsenic and ampicillin sensitive E. coli XL1 blue and environmental strains (Vibrio tubiashii W85 and E. coli W101) were tested for efficiency of uptake of plasmid (P) pUCminusMCS (arsBᴿampᴿ) under varying stress conditions. Transformation experiments revealed that combined effect of carbon, nitrogen and metal on horizontal gene transfer (HGT) was significantly higher (p < 0.01) than individual factors. The effect of carbon on HGT was proved to be superior to nitrogen under metal stressed conditions. Presence of arsenic in experimental setups (P + M, P + N + M and P + C + M) enhanced the HGT compared to non-metal counterparts supplemented with carbon or nitrogen. Arsenic resistant bacterial isolates (n = 200) were tested for the ability to utilize various carbon and nitrogen substrates and distinct positive correlation (p < 0.001) was found between arsenic resistance and utilization of urea and nitrate. However, evident positive correlation was not found between carbon sources and arsenic resistance. Our findings suggest that carbon and nitrogen pollution in aquatic habitats under arsenic stress determine the microbial community dynamics and critically influence uptake of genetic material from the surrounding environment.