Making waves: The NORMAN antibiotic resistant bacteria and resistance genes database (NORMAN ARB&ARG)-An invitation for collaboration to tackle antibiotic resistance.

With the global concerns on antibiotic resistance (AR) as a public health issue, it is pivotal to have data exchange platforms for studies on antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment. For this purpose, the NORMAN Association is hosting the NORMAN ARB&ARG database, which was developed within the European project ANSWER. The present article provides an overview on the database functionalities, the extraction and the contribution of data to the database. In this study, AR data from three studies from China and Nepal were extracted and imported into the NORMAN ARB&ARG in addition to the existing AR data from 11 studies (mainly European studies) on the database. This feasibility study demonstrates how the scientific community can share their data on AR to generate an international evidence base to inform AR mitigation strategies. The open and FAIR data are of high potential relevance for regulatory applications, including the development of emission limit values / environmental quality standards in relation to AR. The growth in sharing of data and analytical methods will foster collaboration on risk management of AR worldwide, and facilitate the harmonization in the effort for identification and surveillance of critical hotspots of AR. The NORMAN ARB&ARG database is publicly available at: https://www.norman-network.com/nds/bacteria/.

Beneficial microbial consortium improves winter rye performance by modulating bacterial communities in the rhizosphere and enhancing plant nutrient acquisition.

The beneficial effect of microbial consortium application on plants is strongly affected by soil conditions, which are influenced by farming practices. The establishment of microbial inoculants in the rhizosphere is a prerequisite for successful plant-microorganism interactions. This study investigated whether a consortium of beneficial microorganisms establishes in the rhizosphere of a winter crop during the vegetation period, including the winter growing season. In addition, we aimed for a better understanding of its effect on plant performance under different farming practices. Winter rye plants grown in a long-time field trial under conventional or organic farming practices were inoculated after plant emergence in autumn with a microbial consortium containing Pseudomonas sp. (RU47), Bacillus atrophaeus (ABi03) and Trichoderma harzianum (OMG16). The density of the microbial inoculants in the rhizosphere and root-associated soil was quantified in autumn and the following spring. Furthermore, the influence of the consortium on plant performance and on the rhizosphere bacterial community assembly was investigated using a multidisciplinary approach. Selective plating showed a high colonization density of individual microorganisms of the consortium in the rhizosphere and root-associated soil of winter rye throughout its early growth cycle. 16S rRNA gene amplicon sequencing showed that the farming practice affected mainly the rhizosphere bacterial communities in autumn and spring. However, the microbial consortium inoculated altered also the bacterial community composition at each sampling time point, especially at the beginning of the new growing season in spring. Inoculation of winter rye with the microbial consortium significantly improved the plant nutrient status and performance especially under organic farming. In summary, the microbial consortium showed sufficient efficacy throughout vegetation dormancy when inoculated in autumn and contributed to better plant performance, indicating the potential of microbe-based solutions in organic farming where nutrient availability is limited.

Long-term patterns of hydrocarbon biodegradation and bacterial community composition in epipelagic and mesopelagic zones of an Arctic fjord.

Oil spill attenuation in Arctic marine environments depends on oil-degrading bacteria. However, the seasonally harsh conditions in the Arctic such as nutrient limitations and sub-zero temperatures limit the activity even for bacteria capable of hydrocarbon metabolism at low temperatures. Here, we investigated whether the variance between epipelagic (seasonal temperature and inorganic nutrient variations) and mesopelagic zone (stable environmental conditions) could limit the growth of oil-degrading bacteria and lead to lower oil biodegradation rates in the epipelagic than in the mesopelagic zone. Therefore, we deployed absorbents coated with three oil types in a SW-Greenland fjord system at 10-20 m (epipelagic) and 615-650 m (mesopelagic) water depth for one year. During this period we monitored the development and succession of the bacterial biofilms colonizing the oil films by 16S rRNA gene amplicon quantification and sequencing, and the progression of oil biodegradation by gas chromatography - mass spectrometry oil fingerprinting analysis. The removal of hydrocarbons was significantly different, with several polycyclic aromatic hydrocarbons showing longer half-life times in the epipelagic than in the mesopelagic zone. Bacterial community composition and density (16S rRNA genes/ cm2) significantly differed between the two zones, with total bacteria reaching to log-fold higher densities (16S rRNA genes/cm2) in the mesopelagic than epipelagic oil-coated absorbents. Consequently, the environmental conditions in the epipelagic zone limited oil biodegradation performance by limiting bacterial growth.

Elevated levels of antibiotic resistance in groundwater during treated wastewater irrigation associated with infiltration and accumulation of antibiotic residues.

Treated wastewater irrigation (TWW) releases antibiotics and antibiotic resistance genes (ARGs) into the environment and might thus promote the dissemination of antibiotic resistance in groundwater (GW). We hypothesized that TWW irrigation increases ARG abundance in GW through two potential mechanisms: the contamination of GW with resistant bacteria and the accumulation of antibiotics in GW. To test this, the GW below a real-scale TWW-irrigated field was sampled for six months. Sampling took place before, during and after high-intensity TWW irrigation. Samples were analysed with 16S rRNA amplicon sequencing, qPCR of six ARGs and the class 1 integron-integrase gene intI1, while liquid chromatography tandem mass spectrometry was performed to detect antibiotic and pharmaceutical residues. Absolute abundance of 16S rRNA in GW decreased rather than increased during long-term irrigation. Also, the relative abundance of TWW-related bacteria did not increase in GW during long-term irrigation. In contrast, long-term TWW irrigation increased the relative abundance of sul1 and intI1 in the GW microbiome. Furthermore, GW contained elevated concentrations of sulfonamide antibiotics, especially sulfamethoxazole, to which sul1 confers resistance. Total sulfonamide concentrations in GW correlated with sul1 relative abundance. Consequently, TWW irrigation promoted sul1 and intI1 dissemination in the GW microbiome, most likely due to the accumulation of drug residues.

Antibiotic resistance gene load and irrigation intensity determine the impact of wastewater irrigation on antimicrobial resistance in the soil microbiome.

Treated wastewater (TWW) irrigation is a useful counter-measure against the depletion of freshwater (FW) resources. However, TWW contains several contaminants of emerging concern, such as antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs). Thus, TWW irrigation might promote the spread of antimicrobial resistance in soil environments. In the present work, we hypothesized that the ARG load and irrigation intensity define the effect of TWW irrigation on ARG spread dynamics in soil. This hypothesis was tested using a multiphase approach: a) comparing soil from a full-scale, commercially operated, TWW irrigated field with non-irrigated soil, b) long-term sampling of the TWW irrigated field over one year with different irrigation intensities and intercepted by irrigation breaks and c) laboratory-scale soil microcosms irrigated with TWW compared to FW. Six ARGs, the integrase gene intI1 and the 16S rRNA were quantified using qPCR. In addition, effects of TWW irrigation on bacterial community composition of microcosm-samples were analysed with 16S rRNA amplicon sequencing. The genes sul1, qnrS, blaOXA-58, tet(M) and intI1 were significantly more abundant in the TWW irrigated field soil, whereas blaCTX--M-32 and blaTEM, the least abundant genes in the TWW irrigation, showed higher abundance in the non-irrigated soil. The relative abundance of sul1, qnrS, blaOXA-58, tet(M) and intI1 correlated with TWW irrigation intensity and decreased during irrigation breaks. Despite the decrease, the levels of these genes remained consistently higher than the non-irrigated soil indicating persistence upon their introduction into the soil. Microcosm experiments verified observations from the field study: TWW irrigation promoted the spread of ARGs and intI1 into soil at far elevated levels compared to FW irrigation. However, the impact of TWW irrigation on 16S rRNA absolute abundance and the soil microbial community composition was negligible. In conclusion, the impact of TWW irrigation depends mainly on the introduced ARG load and the irrigation intensity.

Treated wastewater irrigation promotes the spread of antibiotic resistance into subsoil pore-water.

In the present study, we investigated the impact of treated wastewater (TWW) irrigation on the prevalence of antibiotic resistance genes (ARGs) in subsoil pore-water, a so-far under-appreciated matrix. We hypothesized that TWW irrigation increases ARG prevalence in subsoil pore-water. This hypothesis was tested using a multiphase approach, which consisted of sampling percolated subsoil pore-water from lysimeter-wells of a real-scale TWW-irrigated field, operated for commercial farming practices, and controlled, laboratory microcosms irrigated with freshwater or TWW. We monitored the abundance of six selected ARGs (sul1, blaOXA-58, tetM, qnrS, blaCTX-M-32 and blaTEM), the intI1 gene associated with mobile genetic elements and an indicator for anthropogenic pollution and bacterial abundance (16S rRNA gene) by qPCR. The bacterial load of subsoil pore water was independent of both, irrigation intensity in the field study and irrigation water type in the microcosms. Among the tested genes in the field study, sul1 and intI1 exhibited constantly higher relative abundances. Their abundance was further positively correlated with increasing irrigation intensity. Controlled microcosm experiments verified the observed field study results: the relative abundance of several genes, including sul1 and intI1, increased significantly when irrigating with TWW compared to freshwater irrigation. Overall, TWW irrigation promoted the spread of ARGs and intI1 in the subsoil pore-water, while the bacterial load was maintained. The combined results from the real-scale agricultural field and the controlled lab microcosms indicate that the dissemination of ARGs in various subsurface environments needs to be taken into account during TWW irrigation scenarios.

Evaluation of chemical and biological contaminants of emerging concern in treated wastewater intended for agricultural reuse.

The occurrence of chemical and biological contaminants of emerging concern (CECs) was investigated in treated wastewater intended for reuse in agriculture. An agarose hydrogel diffusion-based passive sampler was exposed to the outlet of a wastewater treatment plant (WWTP) located in Cyprus, which is equipped with membrane bioreactor (MBR). Passive samplers in triplicate were exposed according to a time-series exposure plan with maximum exposure duration of 28 days. Composite flow-proportional wastewater samples were collected in parallel with the passive sampling exposure plan and were processed by solid phase extraction using HORIZON SPE-DEX 4790 and the same sorbent material (Oasis HLB) as in the passive sampler. The analysis of passive samplers and wastewater samples enabled (i) the field-scale calibration of the passive sampler prototype by the calculation of in situ sampling rates of target substances, and (ii) the investigation of in silico predicted transformation products of the four most ecotoxicologically hazardous antibiotics (azithromycin, clarithromycin, erythromycin, ofloxacin). Additionally, the wastewater samples were subjected to the analysis of seven preselected antibiotic resistant genes (ARGs) and one mobile resistant element (int1). All extracts were analyzed for chemicals in a single batch using a highly sensitive method for pharmaceuticals, antibiotics and illicit drugs by liquid chromatography tandem MS/MS (LC-QQQ) and for various other target compounds (2316 compounds in total) by liquid chromatography high-resolution mass spectrometry (LC-HRMS). 279 CECs and all investigated ARGs (except for blaCTX-M-32) were detected, highlighting potential chemical and biological hazards related to wastewater reuse practices. 16 CECs were prioritized following ecotoxicological risk assessment, whereas sul1 and the mobile resistant element (int1) showed the highest abundance. Comprehensive monitoring efforts using novel sampling methods such as passive sampling, wide-scope target screening and molecular analysis are required to assure safe application of wastewater reuse and avoid spread and crop uptake of potentially hazardous chemicals.

Evaluation of a novel quorum quenching strain for MBR biofouling mitigation.

Membrane biofouling, due to Soluble Microbial Products (SMP) and Extracellular Polymeric Substances (EPS) deposition, results in reduction of the performance of Membrane Bioreactors (MBRs). However, recently, a new method of biofouling control has been developed, utilizing the interference of the bacterial inter- and intra-species' communication. Bacteria use Quorum Sensing (QS) to regulate the production of SMP and EPS. Therefore, disruption of Quorum Sensing (Quorum Quenching: QQ), by enzymes or microorganisms, may be a simple mean to control membrane biofouling. In the present study, a novel QQ-bacterium, namely Lactobacillus sp. SBR04MA, was isolated from municipal wastewater sludge and its ability to mitigate biofouling was evaluated by monitoring the changes in critical flux and transmembrane pressure, along with the production of EPS and SMP, in a lab-scale MBR system treating synthetic wastewater. Lactobacillus sp. SBR04MA showed great potential for biofouling control, which was evidenced by the ∼3-fold increase in critical flux (8.3 → 24.25 L/m2/h), as well as by reduction of the SMP and EPS production, which was lower during the QQ-period when compared against the control period. Furthermore, the addition of the QQ-strain did not affect the COD removal rate. Results suggested that Lactobacillus sp. SBR04MA represents a novel and promising strain for biofouling mitigation and enhancement of MBRs performance.

Antibiotic resistance in wastewater treatment plants: Tackling the black box.

Wastewater is among the most important reservoirs of antibiotic resistance in urban environments. The abundance of carbon sources and other nutrients, a variety of possible electron acceptors such as oxygen or nitrate, the presence of particles onto which bacteria can adsorb, or a fairly stable pH and temperature are examples of conditions favouring the remarkable diversity of microorganisms in this peculiar habitat. The wastewater microbiome brings together bacteria of environmental, human and animal origins, many harbouring antibiotic resistance genes (ARGs). Although numerous factors contribute, mostly in a complex interplay, for shaping this microbiome, the effect of specific potential selective pressures such as antimicrobial residues or metals, is supposedly determinant to dictate the fate of antibiotic resistant bacteria (ARB) and ARGs during wastewater treatment. This paper aims to enrich the discussion on the ecology of ARB&ARGs in urban wastewater treatment plants (UWTPs), intending to serve as a guide for wastewater engineers or other professionals, who may be interested in studying or optimizing the wastewater treatment for the removal of ARB&ARGs. Fitting this aim, the paper overviews and discusses: i) aspects of the complexity of the wastewater system and/or treatment that may affect the fate of ARB&ARGs; ii) methods that can be used to explore the resistome, meaning the whole ARB&ARGs, in wastewater habitats; and iii) some frequently asked questions for which are proposed addressing modes. The paper aims at contributing to explore how ARB&ARGs behave in UWTPs having in mind that each plant is a unique system that will probably need a specific procedure to maximize ARB&ARGs removal.