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Domestic wastewater is a significant reservoir of antibiotic resistance genes, which pose environmental and public health risks. We aimed to define an antibiotic resistome signature, represented by core genes, i.e., shared by ≥ 90% of the metagenomes of each of three conceptual environmental compartments – wastewater (influent, sludge, effluent), freshwater, and agricultural soil. The definition of resistome signatures would support the proposal of a framework for monitoring treatment efficacy and assessing the impact of treated wastewater discharge into the environment, such as freshwater and agricultural soil. Metagenomic data from 163 samples originating from wastewater (n = 81), freshwater (n = 58), and agricultural soils (n = 24) across different regions (29 countries, 5 continents), were analysed regarding antibiotic resistance diversity, based on annotation against a database that merged CARD and ResFinder databases. The relative abundance of the total antibiotic resistance genes (corresponding to the ratio between the antibiotic resistance genes and total reads number) was not statistically different between raw and treated wastewater, being significantly higher than in freshwater or agricultural soils. The latter had the significantly lowest relative abundance of antibiotic resistance genes. Genes conferring resistance to aminoglycosides, beta-lactams, and tetracyclines were among the most abundant in wastewater environments, while multidrug resistance was equally distributed across all environments. The wastewater resistome signature included 27 antibiotic resistance genes that were detected in at least 90% of the wastewater resistomes, and that were not frequent in freshwater or agricultural soil resistomes. Among these were genes responsible for resistance to tetracyclines (n = 8), macrolide-lincosamide-streptogramin B (n = 7), aminoglycosides (n = 4), beta-lactams (n = 3), multidrug (n = 2), sulphonamides (n = 2), and polypeptides (n = 1). This comprehensive assessment provides valuable insights into the dynamics of antibiotic resistance in urban wastewater systems and their potential ecological implications in diverse environmental settings. Furthermore, provides guidance for the implementation of One Health monitoring approaches. [Display omitted] • Wastewater, freshwater, and agricultural soil resistomes were screened for shared genes. • Wastewater (influent, sludge, effluent) resistome signature comprised >50 genes. • For freshwater it was not possible to define a signature resistome. • In agricultural soil the resistome signature contained 12 genes. • 27 wastewater resistome signature genes are proposed to chase wastewater impacts. [ABSTRACT FROM AUTHOR]

Abstract The determination of values of abundance of antibiotic resistance genes (ARGs) per mass of soil is extremely useful to assess the potential impacts of relevant sources of antibiotic resistance, such as irrigation with treated wastewater or manure application. Culture-independent methods and, in particular, quantitative PCR (qPCR), have been regarded as suitable approaches for such a purpose. However, it is arguable if these methods are sensitive enough to measure ARGs abundance at levels that may represent a risk for environmental and human health. This study aimed at demonstrating the range of values of ARGs quantification that can be expected based on currently used procedures of DNA extraction and qPCR analyses. The demonstration was based on the use of soil samples spiked with known amounts of wastewater antibiotic resistant bacteria (ARB) (Enterococcus faecalis , Escherichia coli , Acinetobacter johnsonii , or Pseudomonas aeruginosa), harbouring known ARGs, and also on the calculation of expected values determined based on qPCR. The limits of quantification (LOQ) of the ARGs (vanA , qnrS , bla TEM , bla OXA , bla IMP , bla VIM) were observed to be approximately 4 log-units per gram of soil dry weight, irrespective of the type of soil tested. These values were close to the theoretical LOQ values calculated based on currently used DNA extraction methods and qPCR procedures. The observed LOQ values can be considered extremely high to perform an accurate assessment of the impacts of ARGs discharges in soils. A key message is that ARGs accumulation will be noticeable only at very high doses. The assessment of the impacts of ARGs discharges in soils, of associated risks of propagation and potential transmission to humans, must take into consideration this type of evidence, and avoid the simplistic assumption that no detection corresponds to risk absence. Graphical abstract Image 1 Highlights • ARGs limits of quantification and of detection (LOQ & LOD) in soil were investigated. • Soil samples were spiked with ARB harboring known ARGs. • LOQ of vanA , qnrS , bla TEM , bla OXA , bla IMP , bla VIM was >3 log-units and LOD was ∼10 times lower. • High LOQ and LOD values are explained based on intrinsic methodological constraints. • A soil contamination with ∼1000 ARGs copies may be undetected and thus neglected. The results suggest that the limits of quantification and detection of antibiotic resistance genes may be too high to infer about potential risks. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The past decade has witnessed a burst of study regarding antibiotic resistance in the environment, mainly in areas under anthropogenic influence. Therefore, impacts of the contaminant resistome, that is, those related to human activities, are now recognized. However, a key issue refers to the risk of transmission of resistance to humans, for which a quantitative model is urgently needed. This opinion paper makes an overview of some risk-determinant variables and raises questions regarding research needs. A major conclusion is that the risks of transmission of antibiotic resistance from the environment to humans must be managed under the precautionary principle, because it may be too late to act if we wait until we have concrete risk values. [ABSTRACT FROM AUTHOR]

The aim of this study was to assess the fate of invasive antibiotic resistance genes (ARG) discharged in wastewater. With this objective, antibiotic resistant bacteria (ARB) known to harbor specific ARG were inoculated in wastewater (hospital effluent, or municipal raw and treated wastewater) and in ultra-pure sterile water microcosms. Two sets of wastewater ARB isolates were used - set 1, Enterococcus faecalis , Acinetobacter johnsonii , Klebsiella pneumoniae and set 2, Enterococcus faecium , Acinetobacter johnsonii , Escherichia coli . Non-inoculated controls were run in parallel. Samples were collected at the beginning and at the end (15 days) of the incubation period and the abundance of the genes 16S rRNA, int I1, bla TEM and van A and the bacterial community composition were analyzed. In general, the genes bla TEM and van A had lower persistence in wastewater and in ultra-pure water than the genes 16S rRNA or the class 1 integron integrase int I1. This effect was more pronounced in wastewater than in ultra-pure water, evidencing the importance of the autochthonous microbiota on the elimination of invasive ARG. Wastewater autochthonous bacterial groups most correlated with variations of the genes int I1, bla TEM and van A were members of the classes Gammaproteobacteria , Bacilli or Bacteroidia . For bla TEM , but not for van A, the species of the ARB host was important to determine its fate. These are novel findings on the ecology of ARB in wastewater environments. [ABSTRACT FROM AUTHOR]

Demineralized and disinfected waters may have very low microbial loads, requiring that large volumes of water are filtered to recover enough biomass for further analysis. Extended filtration periods, often interrupted by clogging, are a major limiting factor to concentrate samples' microbiota for further examination, besides hindering the work pace. In this study, we investigated the performance of three types of filtering membranes – polycarbonate (PC), cellulose nitrate (CN), and polyethersulfone (PES) with 0.22 µm pore size for culture-independent microbiological analysis (quantitative PCR of seven housekeeping and integrase genes) of tap water, recirculating tap water in a bottle washing loop, and of demineralized water. Compared to PC membranes, CN or PES required lower filtration periods, although had slightly lower DNA extraction yields. However, genes abundance per volume of water was, in general, not significantly different. The exception was observed for bottle washing water in which PC membranes supported significantly higher quantification values than PES membranes. These differences were lower than ∼ 0.5 log-units and did not hamper the distinction of the types of water based on genes profile. Also, the type of membrane did not significantly affect the profile of the bacterial community determined for tap and demineralized water. A major conclusion is that CN membranes, cheaper, allowing shorter filtration periods, and producing results that are not significantly different from those obtained with PC or PES, can be a good alternative to analyze waters with low biomass loads. [Display omitted] • The performance of PC, CN, and PES filters to retain bacterial cells was compared. • Lower vacuum-filtration times were achieved with CN and PES than with PC filters. • DNA yields were slightly lower with CN and PES than with PC filters. • Filter type did not impact gene quantification in clean water samples. • Bacterial community profile was not significantly affected by the filter type. [ABSTRACT FROM AUTHOR]

The environment is the original and most ancient source of the antibiotic resistance determinants that threat the human health nowadays. In the environment, water is a privileged habitat and mode of dissemination of bacteria of different origins. Freshwater bodies that cross urban areas are supposed to hold a complex mixture of both human/animal origin and strictly environmental bacteria. In this study, we were interested in unveiling the bacterial diversity in urban river transects and, simultaneously, investigate the occurrence of antibiotic resistant bacteria, in particular the multidrug resistant (MDR). With this aim, water and sediments of two rivers were sampled from an urban transect and the bacterial diversity was assessed based on 16S rRNA gene-based community analysis and, simultaneously, total heterotrophic bacteria were isolated in the presence and in the absence of antibiotics. The three predominant phyla were Proteobacteria , Bacteroidetes and Actinobacteria , in water, or Acidobacteria , in sediments. MDR bacteria were observed to belong to the predominant phyla observed in water, mostly of the classes Gamma - and Betaproteobacteria ( Proteobacteria ) and Sphingobacteriia and Flavobacteriia ( Bacteroidetes ) and belonged to genera of ubiquitous ( Pseudomonas , Acinetobacter , Stenotrophomonas ) or mainly environmental ( Chitinophaga , Chryseobacterium ) bacteria. The observation that MDR bacteria are widespread in the environment and over distinct phylogenetic lineages has two relevant implications: i) the potential of environmental bacteria as source or facilitators for antibiotic resistance acquisition; ii) the need to complement culture-independent methods with culture-based approaches in order to identify major sources of MDR profiles. [ABSTRACT FROM AUTHOR]

• Emerging microbial challenges call for new analytical methods/approach. • Application of microbial assessment to wastewater disinfection critically reviewed. • Limitations of cultivation methods discussed. • Combination of cultivation and new methods recommended. • Recommendations for UWWTPs managers to evaluate disinfection process efficiency. The development of new methods and technologies for microbial characterization as well as their increasing availability at more affordable costs, has made evident the limitations of the conventional and routinely applied (typically cultivation based) methods to exhaustively and fully characterize the actual effect of disinfection process in urban wastewater treatment plants (UWWTPs). Such problem is even more relevant and of concern if microbial challenges, such as the occurrence of pathogens as well as the spread of antibiotic resistance, are taken into account. Such threats move scientists to investigate new and more effective disinfection processes from one side and new methods, techniques and approaches to characterize disinfection process efficiency from the other side. In this opinion paper, the limitations of routine detection methods are discussed according to the relevant and updated scientific literature to explain how research oriented methods and technologies (namely, quantitative real-time PCR, flow cytometry, metagenetics and metagenomics) can allow a better evaluation of disinfection processes. After a short introduction to the main disinfection processes, the application of different microbial characterization methods is discussed according to distinct challenges, such as pathogens inactivation or antibiotic resistance dissemination, when wastewater safety is of concern (for example in reuse scenarios). The routine and research oriented techniques can be successfully used in complementary way to evaluate disinfection process efficiency. Recommendations for UWWTPs managers for internal control of disinfection process are proposed. [ABSTRACT FROM AUTHOR]

• DNA extraction kit, operator and water samples matrix effects were assessed. • A cell-based DNA internal standard was developed to assess water matrix effect. • Distinct kits resulted in differences on gene quantifications <1 log-unit mL−1. • The matrix effect was irrespective of the water type. • These findings encourage the compilation of qPCR data worldwide. Quantitative PCR (qPCR) has been used to quantify antibiotic resistance genes (ARGs) in water, wastewater, soil, sediment and tissue samples. Concerns regarding the comparability of data obtained in different laboratories has been a major bottleneck to incentivize the compilation of publicly available of ARGs quantifications gathered from different reports. In this study, the influence of the DNA extraction kits (NZY Tissue gDNA Isolation kit or DNeasy PowerWater kit) and of the operator on the DNA extraction yield and on qPCR genes quantification was assessed. Since in wastewater and water samples the matrix effect can affect the DNA recovery and, therefore, gene quantification, an internal standard, consisting in a cloned gene not found in environmental samples, was tested. The aim was to assess how qPCR determinations in wastewater and water samples can be affected by the matrix effect. The results show that the DNA extraction operator did not significantly influence DNA yield. The use of distinct kits resulted in qPCR gene quantifications that did not differ in more than 1 log-unit mL−1. The matrix effect, assessed based on the use of an internal standard, was associated with an underestimation that ranged 0.1–0.9 log gene copy number mL−1 of sample, irrespective of the water type. The reliability on the use of a DNA extraction kit that costs about 3 times less than the most commonly used can be an incentive for the use of DNA based analyses of ARGs in environmental waters. Moreover, the fact that both the DNA extraction operator and the reduced matrix effect have little influence on the final results, are good news, encouraging the compilation of data produced in distinct laboratories. Nevertheless, harmonization efforts are still necessary to minimize bias that may be due associated with other conditions, such as equipment. [ABSTRACT FROM AUTHOR]

• Inter-laboratory quantitative PCR results of effluent-derived ARGs were compared. • The inter-laboratory gene quantification varied from 3% to 28%. • The 16S rRNA gene was the most abundant, followed by intI 1, sul1, qnrS and bla TEM. • The genes bla CTXM-32 and vanA were below the limit of quantification. • Inter-laboratory calibrations increase ARGs quantification reliability. Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are widely distributed in the environment where they represent potential public health threats. Quantitative PCR (qPCR) is a suitable approach to detect and quantify ARGs in environmental samples. However, the comparison of gene quantification data between different laboratories is challenging since the data are predominantly obtained under non-harmonized conditions, using different qPCR protocols. This study aimed at carrying out an inter-laboratory calibration in order to assess the variability inherent to the qPCR procedures for quantification of ARGs. With this aim, samples of treated wastewater collected in three different countries were analysed based on common DNA extract pools and identical protocols as well as distinct equipment, reagents batches, and operators. The genes analysed were the 16S rRNA, vanA , bla TEM , qnrS , sul1 , bla CTXM-32 and intI 1 and the artificial pNORM1 plasmid containing fragments from the seven targeted genes was used as a reference. The 16S rRNA gene was the most abundant, in all the analysed samples, followed by intI 1, sul1, qnrS, and bla TEM , while bla CTXM-32 and vanA were below the limit of quantification in most or all the samples. For the genes 16S rRNA, sul1 , intI1 , bla TEM and qnrS the inter-laboratory variation was below 28% (3–8%, 6–18%, 8–21%, 10–24%, 15–28%, respectively). While it may be difficult to fully harmonize qPCR protocols due to equipment, reagents and operator variations, the inter-laboratory calibration is an adequate and necessary step to increase the reliability of comparative data on ARGs abundance in different environmental compartments and/or geographic regions. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is a global human health threat distributed across humans, animals, plants, and the environment. Under the One-Health concept (humans, animals, and environment), the contamination of water bodies and soil by antibiotic-resistant bacteria cannot be dissociated from its potential transmission to humans. Edible plants can be colonized by a vast diversity of bacteria, representing an important link between the environment and humans in the One-Health triad. Based on multiple examples of bacterial groups that comprise endophytes reported in edible plants, and that have close phylogenetic proximity with human opportunistic pathogens, we argue that plants exposed to human-derived biological contamination may represent a path of transmission of antibiotic resistance to humans. Antibiotic resistance is increasingly disseminated in the environment with an enhanced probability of association with crop plants. Antibiotic-resistant endophytic bacteria, not eliminated during the washing of edible vegetables, might be vectors of transmission of antibiotic resistance to consumers. Crops such as lettuce, carrot, radish, cucumber, or tomato can host antibiotic-resistant endophytic bacteria, potential vectors of transmission to humans. Members of genera such as Enterobacter , Acinetobacter , Pseudomonas , Staphylococcus , Burkholderia , Serratia , Stenotrophomonas , and Bacillus , often associated with crops, may harbor antibiotic-resistance genes. If produced under inadequate conditions and consumed regularly, crops liable to uptake environmental bacteria, may contribute to enrich the acquired antibiotic resistome of consumers. [ABSTRACT FROM AUTHOR]

Drinking water comprises a complex microbiota, in part shaped by the disinfection and distribution systems. Gram-negative bacteria, mainly members of the phylum Proteobacteria , represent the most frequent bacteria in drinking water, and their ubiquity and physiological versatility raises questions about possible implications in human health. The first step to address this concern is the identification and characterization of such bacteria that is the first objective of this study, aiming at identifying ubiquitous or persistent Gram-negative bacteria, Proteobacteria or members of other phyla, isolated from tap water or from its source. > 1000 bacterial isolates were characterized and identified, and a selected group ( n = 68) was further analyzed for the minimum inhibitory concentrations (MIC) to antibiotics (amoxicillin and gentamicin) and metals (copper and arsenite). Total DNA extracts of tap water were examined for the presence of putatively acquired antibiotic resistance or related genes ( intI1 , bla TEM , qnrS and sul1 ). The ubiquitous tap water genera comprised Proteobacteria of the class Alpha- ( Blastomonas , Brevundimonas , Methylobacterium , Sphingobium , Sphingomonas ), Beta- ( Acidovorax , Ralstonia ) and Gamma - ( Acinetobacter and Pseudomonas ). Persistent species were members of genera such as Aeromonas , Enterobacter or Dechloromonas . Ralstonia spp. showed the highest MIC values to gentamicin and Acinetobacter spp. to arsenite. The genes intI1 , bla TEM or sul1 were detected, at densities lower than 2.3 × 10 5 copies/L, 2.4 × 10 4 copies/L and 4.6 × 10 2 copies/L, respectively, in most tap water samples. The presence of some bacterial groups, in particular of Beta- or Gammaproteobacteria (e.g. Ralstonia , Acinetobacter , Pseudomonas ) in drinking water may deserve attention given their potential as reservoirs or carriers of resistance or as opportunistic pathogens. [ABSTRACT FROM AUTHOR]

Members of the genus Aeromonas are recognized carriers of antibiotic resistance in aquatic environments. However, their importance on the spread of resistance from hospital effluents to the environment is poorly understood. Quinolone resistant Aeromonas spp. (n = 112) isolated from hospital effluent (HE) and from raw (RWW) and treated wastewater (TWW) of the receiving urban wastewater treatment plant (UWTP) were characterized. Species identification and genetic intraspecies diversity were assessed based on the 16S rRNA, cpn60 and gyrB genes sequence analysis. The antibiotic resistance phenotypes and genotypes ( qnrA , qnrB , qnrC , qnrD , qnrS , qnrVC ; qepA ; oqxAB ; aac ( 6 ′) -Ib-cr ; bla OXA ; incU) were analyzed in function of the origin and taxonomic group. Most isolates belonged to the species Aeromonas caviae and Aeromonas hydrophila (50% and 41%, respectively). The quinolone and the beta-lactamase resistance genes aac ( 6 ′) -Ib-cr and bla OXA, including gene bla OXA-101 , identified for the first time in Aeromonas spp., were detected in 58% and 56% of the isolates, respectively, with identical prevalence in HE and UWTP wastewater. In contrast, the gene qnrS2 was observed mainly in isolates from the UWTP (51%) and rarely in HE isolates (3%), suggesting that its origin is not the clinical setting. Bacterial groups and genes that allow the identification of major routes of antibiotic resistance dissemination are valuable tools to control this problem. In this study, it was concluded that members of the genus Aeromonas harboring the genes aac ( 6 ′) -Ib-cr and bla OXA are relevant tracers of antibiotic resistance dissemination in wastewater habitats, while those yielding the gene qnrS2 allow the traceability from non-clinical sources. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to infer about the modes and extent of dispersion of Sphingomonadaceae via tap water. Sphingomonadaceae isolated from tap water samples in different places of a hospital were compared, based on intra-species genetic variability and antibiotic resistance phenotypes. These isolates were also compared with others isolated before from houses and dental chairs, served by the same municipal water supply system. Sphingomonadaceae from hospital tap water comprised members of the genera Sphingomonas, Sphingobium, Novosphingobium and Blastomonas. In general, distinct genotypes of Sphingomonadaceae were detected in different hospital areas and in tap water outside the hospital, suggesting these bacteria are not persistent or widespread in the urban water distribution system. Possible intrinsic antibiotic resistance, observed in most or all members of the family or of a genus, was observed for colistin in Sphingomonadaceae, aminoglycosides in the genus Blastomonas and beta-lactams in the genus Sphingobium. Possible acquired resistance phenotypes, not common to all members of a given species, comprised fluoroquinolones, cephalosporins and sulphonamides. Although the potential of Sphingomonadaceae as opportunistic pathogens may be low, the capacity of these bacteria to thrive in water supply systems, combined with the intrinsic or acquired antibiotic resistance, may raise the risk associated with their occurrence in hospital tap water. [Copyright &y& Elsevier]

Abstract: The antibiotic resistance phenotypes of the cultivable bacteria present in nine batches of two Portuguese and one French brands of commercially available mineral waters were examined. Most of the 238 isolates recovered on R2A, Pseudomonas Isolation agar or on these culture media supplemented with amoxicillin or ciprofloxacin, were identified (based on 16S rRNA gene sequence analysis) as Proteobacteria of the divisions Beta, Gamma and Alpha. Bacteria resistant to more than three distinct classes of antibiotics were detected in all the batches of the three water brands in counts up to 102 CFU/ml. In the whole set of isolates, it was observed resistance against all the 22 antimicrobials tested (ATB, bioMérieux and disc diffusion), with most of the bacteria showing resistance to three or more classes of antibiotics. Bacteria with the highest multi-resistance indices were members of the genera Variovorax, Bosea, Ralstonia, Curvibacter, Afipia and Pedobacter. Some of these bacteria are related with confirmed or suspected nosocomial agents. Presumable acquired resistance may be suggested by the observation of bacteria taxonomically related but isolated from different brands, exhibiting distinct antibiotic resistance profiles. Bottled mineral water was confirmed as a possible source of antibiotic resistant bacteria, with the potential to be transmitted to humans. [Copyright &y& Elsevier]

Abstract: Pseudomonas spp. are common inhabitants of aquatic environments, including drinking water. Multi-antibiotic resistance in clinical isolates of P. aeruginosa is widely reported and deeply characterized. However, the information regarding other species and environmental isolates of this genus is scant. This study was designed based on the hypothesis that members of the genus Pseudomonas given their high prevalence, wide distribution in waters and genetic plasticity can be important reservoirs of antibiotic resistance in drinking water. With this aim, the diversity and antibiotic resistance phenotypes of Pseudomonas isolated from different drinking water sources were evaluated. The genotypic diversity analyses were based on six housekeeping genes (16S rRNA, rpoD, rpoB, gyrB, recA and ITS) and on pulsed field gel electrophoresis. Susceptibility to 21 antibiotics of eight classes was tested using the ATB PSE EU (08) and disk diffusion methods. Pseudomonas spp. were isolated from 14 of the 32 sampled sites. A total of 55 non-repetitive isolates were affiliated to twenty species. Although the same species were isolated from different sampling sites, identical genotypes were never observed in distinct types of water (water treatment plant/distribution system, tap water, cup fillers, biofilm, and mineral water). In general, the prevalence of antibiotic resistance was low and often the resistance patterns were related with the species and/or the strain genotype. Resistance to ticarcillin, ticarcillin with clavulanic acid, fosfomycin and cotrimoxazol were the most prevalent (69–84%). No resistance to piperacillin, levofloxacin, ciprofloxacin, tetracycline, gentamicin, tobramycin, amikacin, imipenem or meropenem was observed. This study demonstrates that Pseudomonas spp. are not so widespread in drinking water as commonly assumed. Nevertheless, it suggests that water Pseudomonas can spread acquired antibiotic resistance, preferentially via vertical transmission. [Copyright &y& Elsevier]

Abstract: Since the first commercial cultivation of genetically modified crops in 1994, the rapidly expanding market of genetically modified seeds has given rise to a multibillion dollar industry. This fast growth, fueled by high expectations towards this new commercial technology and shareholder trust in the involved industry, has provided strong incentives for further research and development of new genetically modified plant varieties. Considering, however, the high financial stakes involved, concerns are raised over the influence that conflicts of interest may place upon articles published in peer-reviewed journals that report on health risks or nutritional value of genetically modified food products. In a study involving 94 articles selected through objective criteria, it was found that the existence of either financial or professional conflict of interest was associated to study outcomes that cast genetically modified products in a favorable light (p =0.005). While financial conflict of interest alone did not correlate with research results (p =0.631), a strong association was found between author affiliation to industry (professional conflict of interest) and study outcome (p <0.001). We discuss these results by comparing them to similar studies on conflicts of interest in other areas, such as biomedical sciences, and hypothesize on dynamics that may help explain such connections. [Copyright &y& Elsevier]

Abstract: The species Escherichia coli comprises different subgroups with distinct phylogeny, physiology and ecology and, thus, presumably, with different roles in antimicrobial resistance dissemination. E. coli strains isolated from raw and treated municipal wastewater and from urban water streams were characterized in terms of phylogenetic groups, antimicrobial resistance patterns and the presence of class 1 and class 2 integrons. Our main objective was to investigate the contribution of the different phylo-groups in antimicrobial resistance dissemination in urban waters. Groups A and B1 were predominant in all types of water, evidencing, respectively, the lowest and the highest resistance prevalence. Municipal wastewater treatment was accompanied by significant increases of ciprofloxacin and streptomycin resistance (p<0.01). Antimicrobial resistance prevalence differed significantly between the different phylo-groups and within the same group, mainly in group A. Such differences contributed to explain the higher ciprofloxacin and streptomycin resistance rates observed in treated effluent in comparison with the raw wastewater. We conclude that the dynamics of the bacterial populations has a major role on the dissemination of antimicrobial resistance in the environment. [ABSTRACT FROM AUTHOR]

Abstract: This study was designed to monitor molinate losses in surface and underground waters during Ordram application in a rice field situated in central Portugal. Water samples were collected from different sites, before, during and about one month and a half after Ordram application. Molinate quantification was based on a solid-phase microextraction (SPME) method followed by gas chromatography with flame photometric detector (GC–FPD) analysis, and led to the conclusion that the herbicide was dissipated in the environment, reaching levels as high as 3.9μgl−1 in underground water and 15.8μgl−1 in the river receiving tail waters. The feasibility of the application of treatment methodologies based on adsorption or biodegradation as processes to remove molinate from real-world waters was assessed. These methods seem suitable to reduce molinate concentrations to values in the range of the legally recommended limits (&#60;0.5μgl−1). [Copyright &y& Elsevier]

Ralstonia pickettii are ubiquitous in water environments. Members of this species are frequently, but not always, resistant to both gentamicin and arsenite. Gentamicin and arsenite co-resistance and the putative molecular mechanisms were investigated. A group of 37 R. pickettii strains isolated from drinking water and hospital wastewater were characterized for gentamicin and arsenite resistance phenotypes, the number and size of plasmids, and screened for genetic elements associated with arsenite tolerance, Integrative and Conjugative Elements (ICEs), among other. The genomes of three representative strains were compared. Most gentamicin resistant (GR) isolates (32/33) were resistant to arsenite, and harbored ICE- and ars operon-related genes. These genetic elements were not detected in any of the five arsenite susceptible strains, regardless of the GR (n = 1) or gentamicin susceptibility (GS) (n = 4) phenotype. The comparison of the genomes of two GR (one resistant and one susceptible to arsenite) and one GS strains suggested that these phenotypes correspond to three phylogroups, distinguished by presence of some genes only in GR isolates, in addition to point mutations in functional genes. The presence of ICEs and ars operon-related genes suggest that arsenite resistance might have been acquired by GR lineages. [ABSTRACT FROM AUTHOR]

• Cefotaxime-resistant (CTX-R) coliforms in wastewater were surveyed. • The survey targeted influents and effluents in 57 WWTPs across five continents. • The relative abundance of CTX-R coliforms varied significantly between WWTPs. • Some WWTPs discharged high and potentially hazardous levels of CTX-R coliforms. The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum β-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status. [ABSTRACT FROM AUTHOR]

Abstract Bacteria harboring conjugative plasmids have the potential for spreading antibiotic resistance through horizontal gene transfer. It is described that the selection and dissemination of antibiotic resistance is enhanced by stressors, like metals or antibiotics, which can occur as environmental contaminants. This study aimed at unveiling the composition of the conjugative plasmidome of a hospital effluent multidrug resistant Escherichia coli strain (H1FC54) under different mating conditions. To meet this objective, plasmid pulsed field gel electrophoresis, optical mapping analyses and DNA sequencing were used in combination with phenotype analysis. Strain H1FC54 was observed to harbor five plasmids, three of which were conjugative and two of these, pH1FC54_330 and pH1FC54_140, contained metal and antibiotic resistance genes. Transconjugants obtained in the absence or presence of tellurite (0.5 μM or 5 μM), arsenite (0.5 μM, 5 μM or 15 μM) or ceftazidime (10 mg/L) and selected in the presence of sodium azide (100 mg/L) and tetracycline (16 mg/L) presented distinct phenotypes, associated with the acquisition of different plasmid combinations, including two co-integrate plasmids, of 310 kbp and 517 kbp. The variable composition of the conjugative plasmidome, the formation of co-integrates during conjugation, as well as the transfer of non-transferable plasmids via co-integration, and the possible association between antibiotic, arsenite and tellurite tolerance was demonstrated. These evidences bring interesting insights into the comprehension of the molecular and physiological mechanisms that underlie antibiotic resistance propagation in the environment. Graphical abstract Image 1 Highlights • Conjugative plasmidome analyzed under different conditions, e.g. tellurite or arsenite. • Acquired plasmids conferred new phenotypic and genotypic resistance traits. • Different plasmids and co-integrates were observed in the conjugative plasmidome. • Plasmids of the replicon type HI2 were transferred more frequently than of the type FIA/FIB. • Arsenite 0.5 μM favoured the transfer of plasmid of the replicon type FIA/FIB. [ABSTRACT FROM AUTHOR]

Abstract This research reports for the first time the full-scale application of different homogeneous Advanced Oxidation Processes (AOPs) (H 2 O 2 /UV-C, PMS/UV-C and PMS/Fe(II)/UV-C) for the removal of antibiotics (ABs) and antibiotic resistance genes (ARGs) from wastewater effluent at Estiviel wastewater treatment plant (WWTP) (Toledo, Spain). AOPs based on the photolytic decomposition of H 2 O 2 and peroxymonosulfate tested at low dosages (0.05–0.5 mM) and with very low UV-C contact time (4–18 s) demonstrated to be more efficient than UV-C radiation alone on the removal of the analyzed ABs. PMS (0.5 mM) combined with UV-C (7 s contact time) was the most efficient treatment in terms of AB removal: 7 out of 10 ABs detected in the wastewater were removed more efficiently than using the other oxidants. In terms of ARGs removal efficiency, UV-C alone seemed the most efficient treatment, although H 2 O 2 /UV-C, PMS/UV-C and PMS/Fe(II)/UV-C were supposed to generate higher concentrations of free radicals. The results show that treatments with the highest removal of ABs and ARGs did not coincide, which could be attributed to the competition between DNA and oxidants in the absorption of UV photons, reducing the direct photolysis of the DNA. Whereas the photolytic ABs removal is improved by the generation of hydroxyl and sulfate radicals, the opposite behavior occurs in the case of ARGs. These results suggest that a compromise between ABs and ARGs removal must be achieved in order to optimize wastewater treatment processes. Graphical abstract Unlabelled Image Highlights • Wastewater treatment by Advanced Oxidation Processes studied at full-scale • Removal of antibiotic residues and antibiotic resistance genes assessed • Oxidants photolysis proved more efficient than UV-C alone for antibiotics removal • Sulfate radical based processes did not improve the ARGs removal reached by UV-C • Competition between DNA and oxidants for absorption of UV photons is hypothesized [ABSTRACT FROM AUTHOR]

• Human-associated antibiotic resistance related genes tested as possible biomarkers. • qPCR quantification of 10 biomarkers in raw, treated wastewater and surface water. • Biomarkers' abundance decreased after treatment and dilution in surface water. • Biomarkers can be used for typing of (waste)water quality at global scale. Urban wastewater treatment plants (UWTPs) are essential for reducing the pollutants load and protecting water bodies. However, wastewater catchment areas and UWTPs emit continuously antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with recognized impacts on the downstream environments. Recently, the European Commission recommended to monitor antibiotic resistance in UWTPs serving more than 100 000 population equivalents. Antibiotic resistance monitoring in environmental samples can be challenging. The expected complexity of these systems can jeopardize the interpretation capacity regarding, for instance, wastewater treatment efficiency, impacts of environmental contamination, or risks due to human exposure. Simplified monitoring frameworks will be essential for the successful implementation of analytical procedures, data analysis, and data sharing. This study aimed to test a set of biomarkers representative of ARG contamination, selected based on their frequent human association and, simultaneously, rare presence in pristine environments. In addition to the 16S rRNA gene, ten potential biomarkers (intI1, sul1, ermB, ermF, aph(3′')-Ib, qacEΔ1, uidA, mefC, tetX , and crAssphage) were monitored in DNA extracts (n = 116) from raw wastewater, activated sludge, treated wastewater, and surface water (upstream and downstream of UWTPs) samples collected in the Czech Republic, Denmark, Israel, the Netherlands, and Portugal. Each biomarker was sensitive enough to measure decreases (on average by up to 2.5 log-units gene copy/mL) from raw wastewater to surface water, with variations in the same order of magnitude as for the 16S rRNA gene. The use of the 10 biomarkers allowed the typing of water samples whose origin or quality could be predicted in a blind test. The results show that, based on appropriate biomarkers, qPCR can be used for a cost-effective and technically accessible approach to monitoring wastewater and the downstream environment. [Display omitted] [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]

Although sulfonamide residues are frequently reported as freshwaters contaminants, information on the ability of native bacteria to modify these synthetic antibiotics is scarce. Our purpose was to investigate the potential of bacteria from different aquatic environments to cleave or transform sulfamethoxazole (SMX) and infer on their ability to reduce the toxicity of this antibiotic. From a collection of about 100 Proteobacteria , 47 strains previously isolated from drinking water, surface water, and wastewater grew in the presence of 200 μM SMX , and were further studied. Out of these, 14 strains, mostly from mineral drinking water, transformed SMX into equimolar amounts of the lesser toxic derivative N 4 -acetyl-sulfamethoxazole. The highest percentage of SMX transformation was recorded for two strains affiliated to Pseudomonas mandelii. For P. mandelii McBPA4 higher SMX transformation rate and extent were observed in fed-batch (∼8 μM SMX /h, 81%) than in batch conditions (∼5 μM SMX /h, 25%), but similar specific transformation rates were found in both cultivation modes (∼20 μmol SMX /g cell dry weight /h), indicating the dependence of the process on the microbial load. These results evidence that the capacity to transform synthetic antibiotics may be common among bacteria and highlight the potential of environmental bacteria in attenuating the potential adverse effects of pollution with sulfonamides. [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]

Solar-driven advanced oxidation processes were studied in a pilot-scale photoreactor, as tertiary treatments of effluents from an urban wastewater treatment plant. Solar-H 2 O 2 , heterogeneous photocatalysis (with and/or without the addition of H 2 O 2 and employing three different photocatalysts) and the photo-Fenton process were investigated. Chemical (sulfamethoxazole, carbamazepine, and diclofenac) and biological contaminants (faecal contamination indicators, their antibiotic resistant counterparts, 16S rRNA and antibiotic resistance genes), as well as the whole bacterial community, were characterized. Heterogeneous photocatalysis using TiO 2 -P25 and assisted with H 2 O 2 (P25/H 2 O 2 ) was the most efficient process on the degradation of the chemical organic micropollutants, attaining levels below the limits of quantification in less than 4 h of treatment (corresponding to Q UV  < 40 kJ L −1 ). This performance was followed by the same process without H 2 O 2 , using TiO 2 -P25 or a composite material based on graphene oxide and TiO 2 . Regarding the biological indicators, total faecal coliforms and enterococci and their antibiotic resistant (tetracycline and ciprofloxacin) counterparts were reduced to values close, or beneath, the detection limit (1 CFU 100 mL −1 ) for all treatments employing H 2 O 2 , even upon storage of the treated wastewater for 3-days. Moreover, P25/H 2 O 2 and solar-H 2 O 2 were the most efficient processes in the reduction of the abundance (gene copy number per volume of wastewater) of the analysed genes. However, this reduction was transient for 16S rRNA, intI1 and sul1 genes, since after 3-days storage of the treated wastewater their abundance increased to values close to pre-treatment levels. Similar behaviour was observed for the genes qnrS (using TiO 2 -P25), bla CTX-M and bla TEM (using TiO 2 -P25 and TiO 2 -P25/H 2 O 2 ). Interestingly, higher proportions of sequence reads affiliated to the phylum Proteobacteria ( Beta - and Gammaproteobacteria ) were found after 3-days storage of treated wastewater than before its treatment. Members of the genera Pseudomonas , Rheinheimera and Methylotenera were among those with overgrowth. [ABSTRACT FROM AUTHOR]

The use of reclaimed wastewater (RWW) for the irrigation of crops may result in the continuous exposure of the agricultural environment to antibiotics, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). In recent years, certain evidence indicate that antibiotics and resistance genes may become disseminated in agricultural soils as a result of the amendment with manure and biosolids and irrigation with RWW. Antibiotic residues and other contaminants may undergo sorption/desorption and transformation processes (both biotic and abiotic), and have the potential to affect the soil microbiota. Antibiotics found in the soil pore water (bioavailable fraction) as a result of RWW irrigation may be taken up by crop plants, bioaccumulate within plant tissues and subsequently enter the food webs; potentially resulting in detrimental public health implications. It can be also hypothesized that ARGs can spread among soil and plant-associated bacteria, a fact that may have serious human health implications. The majority of studies dealing with these environmental and social challenges related with the use of RWW for irrigation were conducted under laboratory or using, somehow, controlled conditions. This critical review discusses the state of the art on the fate of antibiotics, ARB and ARGs in agricultural environment where RWW is applied for irrigation. The implications associated with the uptake of antibiotics by plants (uptake mechanisms) and the potential risks to public health are highlighted. Additionally, knowledge gaps as well as challenges and opportunities are addressed, with the aim of boosting future research towards an enhanced understanding of the fate and implications of these contaminants of emerging concern in the agricultural environment. These are key issues in a world where the increasing water scarcity and the continuous appeal of circular economy demand answers for a long-term safe use of RWW for irrigation. [ABSTRACT FROM AUTHOR]

• bla VIM-2 was mostly associated with ST233 and ST111 in Europe. • bla NDM-1 was mostly reported in Asia and distributed by different sequence types. • Significant protein prevalence differences were seen in bla VIM-2 + or bla NDM-1 + accessory genomes. • bla VIM-2 gene frequently inserted in Tn402-like and Tn21 transposons. • bla NDM-1 gene was frequently flanked by ISAba125 or IS91 insertion sequences. Pseudomonas aeruginosa (P. aeruginosa) are ubiquitous opportunistic pathogens that combine intrinsic and acquired multidrug resistance phenotypes. Due to different types of acquired genes, carbapenem resistance has been expanding in this species. This study hypothesised that the spread of carbapenem resistance among P. aeruginosa is influenced by phylogenomic features, being distinct for different genes. To test this hypothesis, the genomes of P. aeruginosa harbouring bla VIM-2 or bla NDM-1 genes were compared. The bla VIM-2 gene was selected because, although frequent, it is almost restricted to this species and bla NDM-1 gene due to its wide interspecies distribution. A group of genomes harbouring the genes bla VIM-2 (n = 116) or bla NDM-1 (n = 27), available in GenBank, was characterised based on core phylogenomic analysis, functional categories in the accessory genome and mobile genetic elements flanking the selected genes. Most bla VIM-2 gene hosts belonged to multilocus sequence types (ST) ST111 (n = 32 of 116) and ST233 (n = 27 of 116) and were reported in Europe (n = 75 of 116). The bla NDM-1 gene hosts were distributed by different STs (ST38, ST773, ST235, ST357 and ST654), frequently from Asia (n = 11 of 27). Significant differences in the prevalence of functional protein/enzyme annotations per number of accessory genomes were observed between bla VIM-2 + and bla NDM-1 +. The bla VIM-2 gene was frequently inserted in the Tn402-like and Tn21 transposons family and rarely in IS6100, while bla NDM-1 gene was preferentially flanked by ISAba125 and ble MBL genes or associated with IS91 insertion sequence. The hypothesis that carbapenem resistance gene acquisition is not random among phylogenomic lineages was confirmed, suggesting the importance of phylogeny in the dissemination of antibiotic resistance genes. [Display omitted] [ABSTRACT FROM AUTHOR]

Disinfection processes aim at reducing the number of viable cells through the generation of damages in different cellular structures and molecules. Since disinfection involves unspecific mechanisms, some microbial populations may be selected due to resilience to treatment and/or to high post-treatment fitness. In this study, the bacterial community composition of secondarily treated urban wastewater and of surface water collected in the intake area of a drinking water treatment plant was compared before and 3-days after disinfection with ultraviolet radiation, ozonation or photocatalytic ozonation. The aim was to assess the dynamics of the bacterial communities during regrowth after disinfection. In all the freshly collected samples, Proteobacteria and Bacteroidetes were the predominant phyla (40–50% and 20–30% of the reads, respectively). Surface water differed from wastewater mainly in the relative abundance of Actinobacteria (17% and < 5% of the reads, respectively). After 3-days storage at light and room temperature, disinfected samples presented a shift of Gammaproteobacteria (from 8 to 10% to 33–65% of the reads) and Betaproteobacteria (from 14 to 20% to 31–37% of the reads), irrespective of the type of water and disinfection process used. Genera such as Pseudomonas, Acinetobacter or Rheinheimera presented a selective advantage after water disinfection. These variations were not observed in the non-disinfected controls. Given the ubiquity and genome plasticity of these bacteria, the results obtained suggest that disinfection processes may have implications on the microbiological quality of the disinfected water. [ABSTRACT FROM AUTHOR]

Photocatalytic ozonation was employed for the first time in continuous mode with TiO 2 -coated glass Raschig rings and light emitting diodes (LEDs) to treat urban wastewater as well as surface water collected from the supply area of a drinking water treatment plant (DWTP). Different levels of contamination and types of contaminants were considered in this work, including chemical priority substances (PSs) and contaminants of emerging concern (CECs), as well as potential human opportunistic antibiotic resistant bacteria and their genes (ARB&ARG). Photocatalytic ozonation was more effective than single ozonation (or even than TiO 2 catalytic ozonation) in the degradation of typical reaction by-products (such as oxalic acid), and more effective than photocatalysis to remove the parent micropollutants determined in urban wastewater. In fact, only fluoxetine, clarithromycin, erythromycin and 17-alpha-ethinylestradiol (EE2) were detected after photocatalytic ozonation, by using solid-phase extraction (SPE) pre-concentration and LC-MS/MS analysis. In surface water, this treatment allowed the removal of all determined micropollutants to levels below the limit of detection (0.01–0.20 ng L −1 ). The efficiency of this process was then assessed based on the capacity to remove different groups of cultivable microorganisms and housekeeping (16S rRNA) and antibiotic resistance or related genes ( intI1, blaTEM , qnrS , sul1 ). Photocatalytic ozonation was observed to efficiently remove microorganisms and ARGs. Although after storage total heterotrophic and ARB (to ciprofloxacin, gentamicin, meropenem), fungi, and the genes 16S rRNA and intI1 , increased to values close to the pre-treatment levels, the ARGs ( blaTEM , qnrS and sul1 ) were reduced to levels below/close to the quantification limit even after 3-days storage of treated surface water or wastewater. Yeast estrogen screen (YES), thiazolyl blue tetrazolium reduction (MTT) and lactate dehydrogenase (LDH) assays were also performed before and after photocatalytic ozonation to evaluate the potential estrogenic activity, the cellular metabolic activity and the cell viability. Compounds with estrogenic effects and significant differences concerning cell viability were not observed in any case. A slight cytotoxicity was only detected for Caco-2 and hCMEC/D3 cell lines after treatment of the urban wastewater, but not for L929 fibroblasts. [ABSTRACT FROM AUTHOR]

Some metals are nowadays considered environmental pollutants. Although some, like Cu and Zn, are essential for microorganisms, at high concentrations they can be toxic or exert selective pressures on bacteria. This study aimed to assess the potential of Cu or Zn as selectors of specific bacterial populations thriving in wastewater. Populations of Escherichia coli recovered on metal-free and metal-supplemented culture medium were compared based on antibiotic resistance phenotype and other traits. In addition, the bacterial groups enriched after successive transfers in metal-supplemented culture medium were identified. At a concentration of 1 mM, Zn produced a stronger inhibitory effect than Cu on the culturability of Enterobacteriaceae . It was suggested that Zn selected populations with increased resistance prevalence to sulfamethoxazole or ciprofloxacin. In non-selective culture media, Zn or Cu selected for mono-species populations of ubiquitous Betaproteobacteria and Flavobacteriia , such as Ralstonia pickettii or Elizabethkingia anophelis , yielding multidrug resistance profiles including resistance against carbapenems and third generation cephalosporins, confirming the potential of Cu or Zn as selectors of antibiotic resistant bacteria. [ABSTRACT FROM AUTHOR]

Abstract: The relationship between antimicrobial residues, antibiotic resistance prevalence and bacterial community composition in hospital effluent and in the receiving wastewater treatment plant was studied. Samples from hospital effluent, raw inflow and final effluent of the receiving wastewater treatment plant were characterized for amoxicillin and ciprofloxacin resistance prevalence, content of heavy metals and antimicrobial residues and bacterial community structure, based on 16S rRNA gene PCR-DGGE analysis. The concentration of fluoroquinolones, arsenic and mercury was in general higher in hospital effluent than in raw inflow, while the opposite was observed for tetracyclines, sulfonamides and penicillin G. The prevalence of ciprofloxacin resistance was significantly higher in hospital effluent than in raw inflow. The concentration of antimicrobial residues was observed to be significantly correlated with the prevalence of antibiotic resistant bacteria and with variations in the bacterial community. Hospital effluent was confirmed as a relevant, although not unique, source of antimicrobial residues and antibiotic resistant bacteria to the wastewater treatment plant. Moreover, given the high loads of antibiotic residues and antibiotic resistant bacteria that may occur in hospital effluents, these wastewater habitats may represent useful models to study and predict the impact of antibiotic residues on bacterial communities. [Copyright &y& Elsevier]

Abstract: Vancomycin is an important antibiotic to treat serious nosocomial enterococci infections. Human activities, in particular those related with clinical practices performed in hospitals, can potentiate the transfer and selection of clinically-relevant resistant bacteria such as vancomycin resistant enterococci (VRE). Indeed, previous studies demonstrated the occurrence of VRE in urban wastewater treatment plants and related environments (e.g. sewage, rivers). In this study, the occurrence of VRE in a hospital effluent and in the receiving urban wastewater treatment plant was investigated. Vancomycin and ciprofloxacin resistant bacteria occurred in the hospital effluent and in raw municipal inflow at densities of 103 to 102 CFUmL−1, being significantly more prevalent in the hospital effluent than in the urban wastewater. Most of the VRE isolated from the hospital effluent belonged to the species Enterococcus faecalis and Enterococcus faecium and presented multidrug-resistance phenotypes to ciprofloxacin, tetracycline, erythromycin, and high-level gentamicin. The same pattern was observed in clinical isolates and in enterococci isolated from the final effluent of the urban wastewater treatment plant. These results show that hospital effluents discharged into urban wastewater treatment plants may be a relevant source of resistance spread to the environment. [Copyright &y& Elsevier]

Abstract: This study was based on the hypothesis that the occurrence of antimicrobial residues and antibiotic resistant bacteria in the sewage could be correlated with the structure and composition of the bacterial community and the antibiotic resistance loads of the final effluent. Raw and treated wastewater composite samples were collected from an urban treatment plant over 14 sampling dates. Samples were characterized for the i) occurrence of tetracyclines, penicillins, sulfonamides, quinolones, triclosan, arsenic, cadmium, lead, chromium and mercury; ii) antibiotic resistance percentages for tetracycline, sulfamethoxazole, ciprofloxacin and amoxicillin and iii) 16S rRNA gene-DGGE patterns. The data of corresponding samples, taking into account the hydraulic residence time, was analyzed using multivariate analysis. Variations on the bacterial community structure of the final effluent were significantly correlated with the occurrence of tetracyclines, penicillins, sulfonamides, quinolones and triclosan in the raw inflow. Members of the class Epsilonproteobacteria presented positive correlations with those antimicrobials, whereas negative correlations were observed with Beta and Gammaproteobacteria and Firmicutes. Antibiotic resistance percentages presented different trends of variation in heterotrophs/enterobacteria and in enterococci, varied over time and after wastewater treatment. Antibiotic resistance was positively correlated with the occurrence of tetracyclines residues and high temperature. A relationship between antibiotic residues, bacterial community structure and composition and antibiotic resistance is demonstrated. Further studies, involving more wastewater treatment plants may help to elucidate this complex relationship. [Copyright &y& Elsevier]

Abstract: Bioaugmentation is reported as a feasible approach for the treatment of pesticide contaminated environments. Aiming the development and future implementation of a bioremediation process to treat natural waters polluted with molinate, a molinate-mineralizing culture, composed of Pseudomonas chlororaphis ON1 and Gulosibacter molinativorax ON4T (designated DC1), was assayed in paddy field floodwater microcosms. The influence of abiotic (temperature, presence of other herbicides) and biotic (floodwater autochthonous microbiota) factors on molinate mineralization by culture DC1 was assessed. In parallel, the proportion of the introduced strains in filter-sterilized floodwaters was monitored by fluorescent in situ hybridization (FISH). Molinate mineralization and growth of culture DC1 were observed under all tested conditions, although the kinetic parameters (maximum specific growth and degradation rates) were significantly affected by the environmental conditions and culture media used. Additionally, these two factors were observed to have a statistically significant interaction. The lowest values of both kinetic parameters were observed at 15 °C. The presence of the herbicides propanil, bentazone and MCPA, frequently applied in rice culture protection, did not affect the degrading capacity of culture DC1. Furthermore, it was possible to infer that the autochthonous microbiota does not retard or limit molinate biodegradation, given the growth and degradation rates by culture DC1 were higher in non-sterile microcosm assays. Although G. molinativorax ON4T is known to promote the initial breakdown of molinate, P. chlororaphis ON1 appeared to be responsible to pull up the process, since higher proportions of this organism were found at the exponential growth and molinate degradation phase. Culture DC1 is efficient, versatile and presents a promising potential to be applied as a bioaugmentation tool for the remediation of environmental waters contaminated with molinate. [Copyright &y& Elsevier]

Abstract: The taxonomic diversity and antibiotic resistance phenotypes of aeromonads were examined in samples from drinking and waste water treatment plants (surface, ground and disinfected water in a drinking water treatment plant, and raw and treated waste water) and tap water. Bacteria identification and intra-species variation were determined based on the analysis of the 16S rRNA, gyrB and cpn60 gene sequences. Resistance phenotypes were determined using the disc diffusion method. Aeromonas veronii prevailed in raw surface water, Aeromonas hydrophyla in ozonated water, and Aeromonas media and Aeromonas puntacta in waste water. No aeromonads were detected in ground water, after the chlorination tank or in tap water. Resistance to ceftazidime or meropenem was detected in isolates from the drinking water treatment plant and waste water isolates were intrinsically resistant to nalidixic acid. Most of the times, quinolone resistance was associated with the gyrA mutation in serine 83. The gene qnrS, but not the genes qnrA, B, C, D or qepA, was detected in both surface and waste water isolates. The gene aac(6’)-ib-cr was detected in different waste water strains isolated in the presence of ciprofloxacin. Both quinolone resistance genes were detected only in the species A. media. This is the first study tracking antimicrobial resistance in aeromonads in drinking, tap and waste water and the importance of these bacteria as vectors of resistance in aquatic environments is discussed. [Copyright &y& Elsevier]

Abstract: Two adjacent paddies of an experimental rice field, subjected to organic and conventional farming, were characterized aiming the comparative assessment of microbiological variations occurring in the bulk paddy soil over the rice cycle. This study comprehended the simultaneous characterization of general physicochemical soil properties [total carbon and nitrogen, pH (H2O and KCl), C:N ratio and water content], biochemical properties [enzymatic activities and Community Level Physiological Profiles (CLPP)], the estimation of cultivable organisms (enumeration of fast growing heterotrophic bacteria, actinomycetes and fungi) and the assessment of bacterial diversity using a culture-independent method (PCR-DGGE fingerprinting). The linkage of the parameters measured was analysed by canonical correspondence analysis (CCA). CCA ordination plots of the CLPP showed a similar pattern of microbial functional activity in both agronomic management systems, except in June. Enzymatic activity, water content and fungi counts were the main factors affecting the observed CLPP time variation. Such a variation was not expressed by the Shannon and evenness indices, which did not evidence significant differences in the bacterial and functional diversity between or within farming type over the analysed period. The cluster and CCA analyses of the DGGE profiles allowed the distinction of the bacterial communities of both paddies, with temporal variations being observed in the organically managed field but not in the conventional paddy. Enzymatic activity, pH and molinate content were the factors which most contributed to the observed variations. Altogether these results underline the functional redundancy of the rice paddy soil and evidence the temporal variations on the metabolic activity of soil, irrespective of farming type. [ABSTRACT FROM AUTHOR]

This study reports the antibiotic resistance patterns of coagulase negative staphylococci (CNS) isolated from a drinking water treatment plant (WTP), a drinking water distribution network, responsible for supplying water to the consumers (WDN), and a wastewater treatment plant (WWTP), responsible for receiving and treating domestic residual effluents. Genotyping and the 16S rRNA gene sequence analysis demonstrated a higher diversity of species both in the WTP (6 species/19 isolates) and WWTP (12 species/47 isolates) than in the WDN (6 species/172 isolates). Staphylococcus pasteuri and Staphylococcus epidermidis prevailed in the WTP and WDN and Staphylococcus saprophyticus in the WWTP. Staphylococci with reduced susceptibility (resistance or intermediary phenotype) to beta-lactams, tetracycline, clindamycin and erythromycin were observed in all types of water and belonged to the three major species groups. The highest resistance rate was found against erythromycin, presumably due to the presence of the efflux pump encoded by the determinant msrA, detected in the majority of the resistant isolates. This study demonstrates that antibiotic resistant CNS may colonize different types of water, namely drinking water fulfilling all the quality standards. [Copyright &y& Elsevier]