The environment is one of the main reservoirs of antibiotic resistance genes (ARGs) but multidrug resistant (MDR) environmental isolates are barely characterised. As suggested by the name, Pedobacter species have been predominantly isolated from soils, but are also recovered from water (including drinking water), chilled food, fish, compost, sludge, glaciers and other extreme environments. The susceptibility phenotype of Pedobacter lusitanus NL19 (isolated from a deactivated uranium mine), its closely related species and the genus type strain were investigated. All strains are MDR bacteria, resistant to β-lactams, colistin, aminoglycosides and ciprofloxacin. Therefore, Pedobacter spp. are likely intrinsically resistant to β-lactams (including ertapenem) and to other three classes of antibiotics. 6%–8% of their total protein-encoding genes encode a diverse collection of putative ARGs, including β-lactamases. These enzymes are highly abundant in all the other Pedobacter strains with sequenced genomes, especially class C, class B3 and class A. LUS-1 and PLN-1 were further characterised in E. coli. LUS-1 is a class A β-lactamase and it conferred an increase in the MIC of cefotaxime, albeit very low. PLN-1 is a class B3 β-lactamase with carbapenemase activity, conferring resistance to ertapenem and a 66x and 16x increase in the MIC of imipenem and meropenem, respectively. PLN-1 also hydrolyses ampicillin, 1st and 3rd generation cephalosporins, and at a lower extent cephamycins and 4th generation cephalosporins. Therefore, Pedobacter spp. encode a large and diverse arsenal of resistance mechanisms that make them environmental superbugs.

Microsorum pteropus is a novel potential Cd (cadmium) aquatic hyperaccumulator. In the present study, hydroponic experiments were conducted to assess the accumulation and subcellular distribution of Cd in the root, stem and leaf of M. pteropus. SEM (scanning electron microscopy) – EDX (energy dispersive X-ray fluorescence spectrometer) and TEM (transmission electron microscopy) were used to observe the ultrastructure of different tissues under 500 μM Cd exposure. After exposure to 500 μM Cd for 7 days, the root, stem and leaf of M. pteropus can accumulate to be > 400 mg/kg Cd in dry mass with no significant influence on the growth. In the root and leaf of M. pteropus, the Cd was more likely to store in the cell wall fraction. However, Cd in the stem was mainly stored in both the cell wall fraction and the cytoplasm fraction. Under SEM observation and EDX detection, 1) Cd was found to be sequestrated in the epidermis or chelated in the root cells, 2) no significant deposit spots were observed in the stem, 3) Cd was found in the trichome of the leaf, and the sporangium was not damaged. TEM observations revealed 1) possible Cd precipitations in the root cell and 2) no significant ultrastructure variation in the stem, and 3) the chloroplast retained its structure and was not affected by the Cd. M. pteropus showed great capacity for Cd accumulation without influencing growth. In addition, the ultrastructure of all the tissues was not damaged by the Cd. M. pteropus showed a great potential in phytoremediation in heavy metal polluted water solutions, and may provide new directions for the study of resistance mechanisms of aquatic hyperaccumulators.

Swine farm and the adjacent farmland are hot spots of ARGs. However, few studies have investigated the on-site occurrence of ARGs distributed in the process of anaerobic digestion (AD) followed by land application of swine wastewater. Two typical swine farms, in southern and northern China respectively, with AD along with land application were explored on ARG distributions. ARGs were highly abundant in raw swine wastewater, AD effectively reduced the copy number of all detected ARGs (0.21–1.34 logs removal), but the relative abundance with different resistance mechanisms showed distinctive variation trends. The reduction efficiency of ARGs was improved by stable operational temperature and longer solid retention time (SRT) of AD. ARGs in soil characterized the contamination from the irrigation of the digested liquor. The total ARGs quantity in soil fell down by 1.66 logs in idle period of winter compared to application period of summer in the northern region, whereas the total amount was steady with whole-year application in south. Some persistent (sul1 and sul2) and elevated ARGs (tetG and ereA) in AD and land application need more attention.

The prevalence of antibiotic resistance and the implicated mechanisms of resistance were evaluated in Enterococcus spp. and Escherichia coli, isolated from a total of 250 faecal samples of echinoderms collected from Azorean waters (Portugal). A total of 144 enterococci (120 Enterococcus faecium, 14 E. hirae, 8 E. faecalis, 2 E. gallinarum) and 10 E. coli were recovered. High percentages of resistance in enterococci were found for erythromycin, ampicillin, tetracyclin and ciprofloxacin. The erm(A) or erm(B), tet(M) and/or tet(L), vat(D), aac(6′)-aph(2″) and aph(3′)-IIIa genes were found in isolates resistant to erythromycin, tetracycline, quinupristin/dalfopristin, high-level gentamicin and high-level kanamycin, respectively. Resistance in E. coli isolates was detected for streptomycin, amikacin, tetracycline and tobramycin. The aadA gene was found in streptomycin-resistant isolates and tet(A)+tet(B) genes in tetracycline-resistant isolates. The data recovered are essential to improve knowledge about the dissemination of resistant strains through marine ecosystems and the possible implications involved in transferring these resistances either to other animals or to humans.

Antibiotic resistance in bacteria is a growing problem that is not only restricted to the clinical setting but also to other environments such as marine species that harbor antibiotic resistant bacteria and therefore may serve as reservoirs for antibiotic-resistance genetic determinants. The aim of this study was to evaluate antibiotic resistance phenotypes in enterococci isolated from fecal samples of gilthead seabream and the associated mechanisms of resistance. A collection of 118 samples were analyzed and 73 enterococci were recovered. The strains showed high percentages of resistance to erythromycin and tetracycline (58.9% and 17.8%, respectively). Lower level of resistance (<13%) was detected for quinupristin–dalfopristin, ampicillin, high-level-gentamicin, high-level-streptomycin, high-level-kanamycin, ciprofloxacin and chloramphenicol. The erm(B), tet(L) or tet(M), aac(6′)-aph(2″) and aph(3′)-IIIa genes were shown in isolates resistant to erythromycin, tetracycline, high-level gentamicin and high-level kanamycin, respectively. Antibiotic resistance in natural microbiota is becoming a concern of human and environmental health.

A dose–response study was performed in four commercial clones, Baldo (Populus deltoides), Jean Pourtet (Populus nigra), I-214 (Populus x euramericana) and Villafranca (Populus alba), to investigate the best performing species in terms of metal content and high metal resistance (absence of symptoms) useful in biomass production on contaminated water/land by zinc. Zinc (1 μM as control and 1 mM) was applied for 4 weeks in a hydroponic system. Clone Villafranca was the least damaged, while the most sensitive was clone I-214. The highest zinc concentration in all different parts of plants analysed was recorded in Villafranca > I-214 > Baldo > Jean Pourtet. The higher translocation factor was seen in Baldo, the lowest in Villafranca. Analyses of leaf damage showed a reduction on Chl a in young leaves (96 %) in I-214 stressed plants, whereas in Villafranca, Chl a was about double compared to the control. Regarding other photosynthetic pigments, violaxanthin was significantly correlated to zinc concentration in old leaves. The responses of clones to zinc (Zn) stress were specific: Villafranca was the most resistant, while I-214 showed the highest biomass production under Zn excess. Since these two clones have useful and complementary traits for uptake and detoxification while maintaining high biomass production under Zn excess, they are interesting candidates for understanding the key resistance mechanisms.

The medium most directly affected by anthropic contamination is soil and, hence, groundwater (saturated and unsaturated zones). In the phytoremediation process, the direct absorption of soil contaminants through the roots is a surprising pollutant removal mechanism. Plants can act as a natural filter of shallow groundwater contamination, controlling and reducing the vertical percolation of contaminants into the soil, and after reaching the level of the water table, the roots can absorb contaminants dissolved in the water, thus reducing the size of the plume and protecting receptor sites (water supply wells, rivers, lakes) from possible contamination. In the first phase of the research, assays were performed to evaluate the tolerance of plant species to the direct injection of a benzene solution into the roots. Subsequent experiments involved direct absorption and spraying. The aim of this study was to evaluate the potential for tolerance and reaction to high levels of benzene. Three plant species were used, an herbaceous ornamental plant (Impatiens walleriana), a fern (Pteris vittata), and forage grass (Brachiaria brizantha). At the end of the study, the surface changes caused by VOCs (aerial structures) of benzene were evaluated, using an environmental scanning electron microscope (ESEM) to identify possible mechanisms of resistance of the plant to air pollution, i.e., hydrocarbon pollution. The plant material used here was young plant species selected for study. For the analysis by gas chromatography (GC), the plant material was separated into aerial (stem, leaves, and flowers) and underground parts (roots). A comparison of the benzene content in different parts of the plant indicated a higher concentration in the stem + leaves, followed by the roots, which is justified by its translocation inside the plant. P. vittata showed low uptake (5.88 %) mainly in the root and (<2 %) in the leaves, which was also observed in the tolerance experiment, in which visual symptoms of toxicity were not observed. I. walleriana showed benzene removal rates of approximately 18.7 % (injection into the soil) as a result of direct absorption through the roots. After the treatment was suspended, I. walleriana gradually reacted to the detoxification process by recovering its stem stiffness and normal color. B. brizantha showed intermediate behavior and did not react to the detoxification process.

Constanta harbor has been contaminated for decades with petroleum and petroleum products, which contain different toxic organic solvents. A novel solvent-tolerant bacterium, Vibrio alginolyticus IBBCₜ₂ was isolated from a seawater sample (Constanta harbor). Alkanes (i.e., n-hexane, n-decane, cyclohexane) with logarithm of partition coefficient in n-octanol and water (log P OW ) > 3.35, were less toxic for V. alginolyticus strain IBBCₜ₂, compared with aromatics (i.e., toluene, m-xylene, ethylbenzene) with log P OW < 3.17. The high organic solvent resistance of V. alginolyticus IBBCₜ₂ could be due to the presence of some catabolic (alkB, alkB/alkB1, todC1, xylM, C23DO) and transporter (HAE1, acrAB) genes. The adaptation mechanisms, underlying cyclohexane, n-hexane, n-decane, toluene, m-xylene, and ethylbenzene resistance in V. alginolyticus IBBCₜ₂ showed a complex response of cells 60 min after solvent shock (i.e., modifications of the cell viability, changes in the membrane’s lipid and protein profile, modifications of the genomic fingerprinting). Exposure of V. alginolyticus IBBCₜ₂ cells to salt stress decreases the organic solvents tolerance of this bacterium.

Soil that is co-contaminated with heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) is difficult to bioremediate due to the ability of toxic metals to inhibit PAH degradation by bacteria. We demonstrated the resistance mechanisms to Cu(II) and Cd(II) of two newly isolated strains of Sphingobium sp. PHE-SPH and Ochrobactrum sp. PHE-OCH and further tested their potential application in the bioremediation of HM-phenanthrene (PhA) co-contaminated sites. The PHE-SPH and PHE-OCH strains tolerated 4.63 and 4.34 mM Cu(II) and also showed tolerance to 0.48 and 1.52 mM Cd(II), respectively. Diverse resistance patterns were detected between the two strains. In PHE-OCH cells, the maximum accumulation of Cu(II) occurred in the cell wall, while the maximum accumulation was in the cytoplasm of PHE-SPH cells. This resulted in a sudden suppression of growth in PHE-OCH and a gradual inhibition in PHE-SPH as the concentration of Cu(II) increased. Organic acid production was markedly higher in PHE-OCH than in PHE-SPH, which may also have a role in the resistance mechanisms, and contributes to the higher Cd(II) tolerance of PHE-OCH. The factors involved in the absorption of Cu(II) or Cd(II) in PHE-SPH and PHE-OCH were identified as proteins and carbohydrates by Fourier transform infrared (FT-IR) spectroscopy. Furthermore, both strains showed the ability to efficiently degrade PhA and maintained this high degradation efficiency under HM stress. The high tolerance to HMs and the PhA degradation capacity make Sphingobium sp. PHE-SPH and Ochrobactrum sp. PHE-OCH excellent candidate organisms for the bioremediation of HM-PhA co-contaminated sites.

Background, aim, and scope In this work, the potential for using olive-mill solid waste as an organic amendment for biochemical and biological restoration of a trichloroethylene-contaminated soil, which has previously been stabilized through vermicomposting processes, has been explored. Materials and methods Trichloroethylene-contaminated water was pumped into soil columns with a layer of vermicompost at 10-cm depth (biobarrier system). The impacts of the trichloroethylene on the microbial community were evaluated by determining: (1) the overall microbial activity (estimated as dehydrogenase activity) and enzyme activities related to the main nutrient cycles (β-glucosidase, o-diphenoloxidase, phosphatase, urease, and arylsulphatase activities). In addition, isoelectric focusing of the soil extracellular humic-β-glucosidase complexes was performed to study the enzymatically active humic matter related to the soil carbon cycle. (2) The soil bacterial diversity and the molecular mechanisms for the bacterial resistance to organic solvents were also determined. For this, polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) was used to detect changes in bacterial community structure and PCR-single-strand conformational polymorphism (SSCP) was developed and optimised for detection and discrimination of the resistance-nodulation-division (RND) genes amplified from the contaminated soils. Results Vermicompost reduced, with respect to the unamended soil, about 30% of the trichloroethylene leaching during the first month of the experiment. Trichloroethylene had a marked negative effect on soil dehydrogenase, β-glucosidase, urease, phosphatase, and arylsulphatase activities. Nevertheless, the vermicompost tended to avoid this toxic effect. Vermicompost also displays stable humic-β-glucosidase complexes that increased the extracellular activity related to C-cycle in the contaminated soils. The isoelectric focusing technique showed a more biochemically active humic matter in the soil sampled under the vermicompost. The behaviour of the three main phyla of bacteria isolated from the DGGE bands was quite different. Bands corresponding to Actinobacteria disappeared, whereas those affiliated with Proteobacteria remained after the trichloroethylene contamination. The disappeared Actinobacteria became visible in the soil amended with the vermicompost. Bands corresponding to Bacteriodetes appeared only in columns of contaminated soils. In this study, six types of RND proteins were detected by PCR-SSCP in the natural soil, three in the trichloroethylene-contaminated soil and 7/5 in trichloroethylene-contaminated soil above/below the vermicompost in the biobarrier columns. Trichloroethylene tended to reduce or eliminate all the clones detected in the uncontaminated soil, whereas new efflux pumps appeared in the biobarrier columns. Discussion Although enzymes incorporated into the humic substances of vermicomposted olive wastes are quite stable, trichloroethylene also inhibited the background levels of the soil extracellular β-glucosidase activity in the amended soils. The decrease was less severe in the biobarrier system, but in any case, no relation was found between the levels of trichloroethylene in soil and extracellular β-glucosidase activity, or between the latter and the quantity of humic carbon in soils. The isoelectric focusing technique was carried out in the humic fraction to determine whether the loss of activity occurred in overall extracellular β-glucosidase or in that linked to stable humic substances (humic-enzyme complexes). The contaminated soils showed the lower enzyme activities, whereas contaminated and amended soils presented greater quantity of focalised (and therefore stable) humic carbon and spectra heterogeneity: very different bands with higher enzyme activities. No clear relationship between trichloroethylene concentration in soil and diversity of the bacterial population was noted. Similar patterns could be found when the community structures of bacteria and microbial activity were considered. Since the use of the dehydrogenase assay has been recognised as a useful indicator of the overall measure of the intensity of microbial metabolism, these results could be attributed to PCR-DGGE methodology, since the method reveals the presence of dominant populations regardless of their metabolic state. Trichloroethylene maintained or even increased the number of clones with the DNA encoding for RND proteins, except for the contaminated soil located above the vermicompost. However, the main effect of trichloroethylene was to modify the structure of the community in contaminated soils, considering the type of efflux pumps encoded by the DNA extracted from soil bacteria. Conclusions Trichloroethylene inhibited specific functions in soil and had a clear influence on the structure of the autochthonous bacterial community. The organic matter released by the vermicomposted olive waste tended to avoid the toxic effect of the contaminant. Trichloroethylene also inhibited the background levels of the soil extracellular β-glucosidase activity, even when vermicompost was present. In this case, the effect of the vermicompost was to provide and/or to stimulate the humic-β-glucosidase complexes located in the soil humic fraction >10⁴, increasing the resistance of the enzyme to the inhibition. The bacterial community from the soil presented significantly different mechanisms to resistance to solvents (RND proteins) under trichloroethylene conditions. The effect of the vermicompost was to induce these mechanisms in the autochthonous bacterial community and/or incorporated new bacterial species, able to grow in a trichloroethylene-contaminated ambient. Coupled biochemical and molecular methodologies are therefore helpful approaches in assessing the effect of an organic amendment on the biochemical and biological restoration of a trichloroethylene-contaminated soil. Recommendations and perspectives Since the main biochemical and biological effects of the organic amendment on the contaminated soil seem to be the incorporation of biochemically active humic matter, as well as new bacterial species able to grow in a trichloroethylene-contaminated ambient, isoelectric focusing and PCR-SSCP methodologies should be considered as parts of an integrated approach to determine the success of a restoration scheme.